Tooth Whitening Compositions and Methods

ABSTRACT

A non-cytotoxic chlorine dioxide composition and a mixed agent bleaching composition for tooth whitening are disclosed. Methods of use are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit pursuant to 35 U.S.C. §119(e) ofU.S. Provisional Application Nos. 61/135,011, filed on Jul. 15, 2008;61/106,026, filed Oct. 16, 2008; and 61/150,685, filed Feb. 6, 2009,each of which is hereby incorporated by reference in its entiretyherein.

BACKGROUND

Efforts to whiten teeth have a long history, thought to date back to theAncient Egyptians. Modem science has provided a detailed understandingof the factors that contribute to tooth color, which has enabledimproved products and methods for whitening. The normal shade of teethis determined by the natural off-white tints of the enamel and thedentin beneath. Extrinsic and intrinsic staining also contribute totooth.

Extrinsic staining refers to surface stains, such as those caused bytea, coffee, red wine, and other foods rich in polyphones. Extrinsicstaining primarily occurs as a result of charged surface interactionsbetween the positively-charged food molecules and the negatively-chargedtooth pellicle, the protein film on the tooth surface that is derivedfrom salivary proteins. Extrinsic stains are removed through the use ofsurfactants and/or abrasives, which cause their physical removal fromthe tooth surface.

Intrinsic staining refers to stains that exist below enamel surface, orthat penetrate below enamel surface. Intrinsic staining can happen whenfood molecules seep into enamel flaws and cracks, or, in some cases,between enamel rods. Intrinsic discoloration can also occur following achange to the structural composition or thickness of the dental hardtissues. Certain metabolic diseases and tooth trauma can also causeintrinsic staining. Tetracycline also causes intrinsic staining.

Removal of intrinsic staining is more difficult and time consuming thanremoval of extrinsic staining. Intrinsic stain removal can be achievedby a variety of methods including use of peroxides or peroxide analogs,with or without chemical, light or heat activation, to bleach thestains. This method oxidizes organic compounds within the enamel anddentin, thereby changing colored materials to non-colored materials; itdoes not remove the stain itself. Acids and dehydration methods, whichlead to opacification of enamel to obscure the subsurface stains, arealso used to remove or mask intrinsic staining.

Tooth whitening products are available over-the-counter and asprofessional services in a dentist's office. Over-the-counter productstypically contain carbamide peroxide or hydrogen peroxide as thebleaching agent. These products have concentrations of up to 21%carbamide peroxide (equivalent to 7% hydrogen peroxide) or as much as10% hydrogen peroxide. They also contain carbomers (for thickening andcontrol) and acidifiers (for peroxide stabilization in aqueoussolution), or alternatively have an anhydrous glycerin base. In-officetreatments almost always use hydrogen peroxide as the oxidizer, atconcentrations of 15% or more, and typically in the 25 to 35% range. Atthese high concentrations, rubber dams, or liquid dams with propersuction, must be used to prevent gingival irritation and ingestion.Additionally, due to their high strengths, products for professionaltreatment require more thickener and more acidification to make themstable, compared to home-use products. Furthermore, professionalchair-side formulas have secondary and often even tertiary andquaternary activators to stimulate a more rapid result. These activatorstake the form of pH modifiers, light sources, and heat sources.

Tooth whitening products, both over-the counter and particularlyprofessional treatments, have several unpleasant side effects, includingtooth sensitivity, soft tissue irritation and tooth surface changes.

Transient tooth sensitivity is the most common side effect reported.Hydrogen peroxide and carbamide peroxide have not been found to inducepathological pulpal changes in testing, although 10% carbamide peroxidehas been reported as causing mild, reversible histological changes. Itis believed that the hypersensitivity associated with whitening iscaused by dehydration, due to the acidified and thickened, substantiallyanhydrous, hydrophillic gels used in the peroxide formulations and thatare held against the teeth. Dehydration results in a negative osmoticpressure and in odontoblastic processes being drawn into the dentinaltubules. Other factors that may contribute to dehydration includewhitening lights used in in-office treatments. While sensitivity may betransient, it is a very undesirable side effect.

Oral mucosa irritation is the second most-common side effect reported.Systems using higher concentrations of hydrogen peroxide or carbamideperoxide result in more gingival irritation than lower concentrationformulations. While peroxide is regarded as safe at low concentrations,peroxide has the potential to induce cell changes at high concentrationsover an extended period of time.

Tooth surface changes have been observed for hydrogen peroxide andcarbamide peroxide in in-vitro testing. Although recent reports on thisissue have conflicting results, this aspect of tooth whitening productscurrently available remains a point of concern. In addition, toothwhitening formulations are usually acidic; acids can decalcify and etchteeth, causing a temporary opacification of underlying discolorations.These side effects often result in the need for remineralizationtherapies in connection with tooth whitening treatment, particularlythose using professional products.

Another problem with current tooth whitening formulations is rebound.Rebound is the phenomenon wherein stains re-appear after a relativelyshort period of time after tooth whitening. The time that elapsespost-treatment before this occurs varies from a few days to weeks, whileother studies claim up to 47 months before any rebound effect occurs(Leonard et al., 2001, J. Esthet. Restor. Dent. 13(6): 357-369). Onestudy found a rebound in 40% of patients at six months with use ofconcentrations ranging from 16%-18% carbamide peroxide (Brunton et al.,2004, Oper. Dent. 29(6): 623-626).

Chlorine dioxide (ClO₂) has been suggested as an alternative to peroxidebased bleaching agents for tooth whitening applications. Chlorinedioxide (ClO₂) is well known as a disinfectant as well as a strongoxidizing agent. The bactericidal, algaecidal, fungicidal, bleaching anddeodorizing properties of chlorine dioxide are also well known.

Chlorine dioxide (ClO₂) is a neutral compound of chlorine in the +IVoxidation state. It disinfects by oxidation; however, it does notchlorinate. It is a relatively small, volatile, and highly energeticmolecule, and a free radical even in dilute aqueous solutions. Chlorinedioxide functions as a highly selective oxidant due to its unique,one-electron transfer mechanism in which it is reduced to chlorite (ClO₂⁻). The pKa for the chlorite ion/chlorous acid equilibrium, is extremelylow at pH 1.8. This is remarkably different from the hypochlorousacid/hypochlorite base ion pair equilibrium found near neutrality, andindicates that the chlorite ion will exist as the dominant species indrinking water.

One of the most important physical properties of chlorine dioxide is itshigh solubility in water, particularly in chilled water. In contrast tothe hydrolysis of chlorine gas in water, chlorine dioxide in water doesnot hydrolyze to any appreciable extent but remains in solution as adissolved gas.

The traditional method for preparing chlorine dioxide involves reactingsodium chlorite with gaseous chlorine (Cl₂(g)), hypochlorous acid(HOCl), or hydrochloric acid (HCl). The reactions are:

2NaClO₂+Cl₂(g)=2ClO₂(g)+2NaCl   [1a]

2NaClO₂+HOCl=2ClO₂(g)+NaCl+NaOH   [1b]

5NaClO₂+4HCl=4ClO₂(g)+5NaCl+2H₂O   [1c]

Reactions [1a] and [1b] proceed at much greater rates in acidic medium,so substantially all traditional chlorine dioxide generation chemistryresults in an acidic product solution having a pH below 3.5. Also,because the kinetics of chlorine dioxide formation are high order inchlorite anion concentration, chlorine dioxide generation is generallydone at high concentration (>1000 ppm), which must be diluted to the useconcentration for application.

Chlorine dioxide may also be prepared from chlorate anion by eitheracidification or a combination of acidification and reduction. Examplesof such reactions include:

2NaClO₃+4HCl→2ClO₂+Cl₂+2H₂O+2NaCl   [2a]

2HClO₃+H₂C₂O₄→2ClO₂+2CO₂+2H₂O   [2b]

2NaClO₃+H₂SO₄+SO₂→2ClO₂+2NaHSO₄   [2c]

At ambient conditions, all reactions require strongly acidic conditions;most commonly in the range of 7-9 N. Heating of the reagents to highertemperature and continuous removal of chlorine dioxide from the productsolution can reduce the acidity needed to less than 1 N.

A method of preparing chlorine dioxide in situ uses a solution referredto as “stabilized chlorine dioxide.” Stabilized chlorine dioxidesolutions contain little or no chlorine dioxide, but rather, consistsubstantially of sodium chlorite at neutral or slightly alkaline pH.Addition of an acid to the sodium chlorite solution activates the sodiumchlorite, and chlorine dioxide is generated in situ in the solution. Theresulting solution is acidic. Typically, the extent of sodium chloriteconversion to chlorine dioxide is low and a substantial quantity ofsodium chlorite remains in the solution.

U.S. Pat. No. 6,582,682 discloses an oral care composition comprising“stabilized chlorine dioxide” that, upon exposure to the mildly acidicpH in the oral cavity, produces chlorine dioxide.

U.S. Pat. No. 6,479,037 discloses preparing a chlorine dioxidecomposition for tooth whitening wherein the composition is prepared bycombining a chlorine dioxide precursor (CDP) portion with an acidulant(ACD) portion. The CDP portion is a solution of metal chlorite at a pHgreater than 7. The ACD is acidic, preferably having a pH of 3.0 to 4.5.The CDP is applied to the tooth surface. The ACD is then applied overthe CDP to activate the metal chlorite and produce chlorine dioxide. ThepH at the contact interface is preferably less than 6 and, mostpreferably, in the range of about 3.0 to 4.5. Thus, the resultingchlorine dioxide composition on the tooth surface is acidic.Additionally, this method exposes the oral mucosa to possible contactwith a strongly highly acidic reagent (ACD).

There is a need in the art for tooth whitening compositions and methodswith reduced side effects.

SUMMARY

The following embodiments meet and address these needs. The followingsummary is not an extensive overview of the embodiment. It is intendedto neither identify key or critical elements of the various embodimentsnor delineate the scope of them.

In one aspect, a bleaching composition for whitening a tooth surface isprovided. The bleaching composition comprises chlorine dioxide and atleast a second bleaching agent, wherein: i) the cytotoxicity of thebleaching composition is about equal to or less than the cytotoxicity ofa composition comprising no chlorine dioxide and a sufficient quantityof the at least second bleaching agent to have comparable bleachingefficacy; ii) the bleaching composition is associated with soft oraltissue irritation about equal to or less irritation than associated witha composition comprising no chlorine dioxide and a sufficient quantityof the at least second bleaching agent to have comparable bleachingefficacy; or iii) the bleaching composition is associated with hardtooth tissue damage about equal to or less than the damage associatedwith a composition comprising no chlorine dioxide and a sufficientquantity of the at least second bleaching agent to have comparablebleaching efficacy. In some embodiments, the at least second bleachingagent can be selected from the group consisting of: alkali metalpercarbonates, carbamide peroxide, sodium perborate, potassiumpersulfate, calcium peroxide, zinc peroxide, magnesium peroxide,hydrogen peroxide complexes, hydrogen peroxide, and combinationsthereof. In some embodiments, the composition has a bleaching efficacy,and the bleaching efficacy is synergistic relative to the efficacies ofthe individual bleaching agents.

The composition can comprise about 5 to about 1000 ppm chlorine dioxidein some embodiments. In an embodiment, the composition comprises about30 to about 40 ppm chlorine dioxide. In some embodiments, thecomposition comprises less than about 0.2 milligrams oxy-chlorine anionper gram composition. In some embodiments, the composition has a pH fromabout 4.5 to about 11. In other embodiments, the composition has a pHfrom about 5 to about 9, or a pH greater than about 6 and less thanabout 8. In an embodiment, the composition has a pH equal to or lessthan about pH 5 and the composition further comprises a remineralizingagent. Optionally, the composition further comprises a desensitizingagent.

Optionally, in some embodiments, the bleaching composition comprises athickener component. The thickener component can be selected from thegroup consisting of natural hydrocolloids, semisynthetic hydrocolloids,synthetic hydrocolloids, and clay. In some embodiments, the thickenercomponent can be a semisynthetic hydrocolloid. An exemplarysemisynthetic hydrocolloid is carboxymethylcellulose.

In another aspect, a method of whitening a tooth surface using thebleaching composition is provided. The method comprises contacting asurface of a tooth with an efficacious amount of the bleachingcomposition. In some embodiments, the contacted tooth surface iswhitened by at least about 3 shade value units. In other embodiments,the contacted tooth surface is whitened by at least about 6 shade valueunits. All embodiments of the bleaching composition are contemplated foruse in the method.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the compositions, kits, and methods,there are depicted in the drawings certain embodiments. However, thecompositions, kits and methods of their use are not limited to theprecise arrangements and instrumentalities of the embodiments depictedin the drawings.

FIG. 1 is a bar graph depicting tooth whitening data for a non-cytotoxicClO₂-containing composition and a commercial over-the-counter (OTC)product having 10% hydrogen peroxide as a function of total treatmenttime. ClO₂=data for non-cytotoxic ClO₂-containing composition. OTC=datafor commercial product having 10% hydrogen peroxide.

FIG. 2 is a graph depicting tooth whitening data for a non-cytotoxicClO₂-containing composition in comparison to a professional whiteninggel comprising 36% hydrogen peroxide as the bleaching agent.

FIG. 3A-3C are a series of representative scanning electron microscopy(SEM) photomicrography images of enamel surface at 2500× magnification.FIG. 3A is the enamel of an untreated tooth. FIG. 3B is enamel surfaceafter treatment with a non-cytotoxic ClO₂-containing composition. FIG.3C is enamel surface after treatment with a professional whitening gelcontaining 36% hydrogen peroxide.

FIGS. 4A-4C are a series of representative SEM microphotograph images ofdentin surface at 5000× magnification. FIG. 4A is the dentin of anuntreated tooth. FIG. 4B is dentin surface after treatment with an OTCwhitening gel containing 10% hydrogen peroxide. FIG. 4C is dentinsurface after treatment with a non-cytotoxic ClO₂-containingcomposition.

DETAILED DESCRIPTION

Provided is a method of whitening a tooth using a composition comprisinga bleaching agent, wherein the composition is not substantiallycytotoxic. In a preferred embodiment, the bleaching agent is chlorinedioxide.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art. Generally, the nomenclature used herein andthe laboratory procedures in cytopathicity analysis, microbial analysis,organic and inorganic chemistry, and dental clinical research are thosewell known and commonly employed in the art.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which it is used.Generally, “about” encompasses a range of values that are plus/minus 10%of a reference value. For instance, “about 25%” encompasses values from22.5% to 27.5%.

It is understood that any and all whole or partial integers between anyranges set forth herein are included herein.

As used herein, “biocidal” refers to the property of inactivating orkilling pathogens, such as bacteria, algae and fungi.

As used herein, “NaDCCA” refers to sodium dichloroisocyanurate and/orthe dihydrate thereof.

As used herein, “tooth whitening” refers to a lightening of tooth shaderelative to the tooth shade prior to treatment. Lightening is assessedon an isolated or an in situ tooth by standard, art-recognized methodsof assessing tooth shade, which include qualitative, quantitative andsemi-quantitative methods. For instance, lightening may be assessed bysimple visual inspection, e.g., by comparing “before” and “after”photographs of the treated teeth. Alternatively, a tooth may be deemedwhitened when the tooth shade relative to the tooth shade prior totreatment is two or more shades lighter, as assessed by Vita classicalshade guide (preferably, under controlled visible light conditions) ortwo or more levels as assessed using the Vita Bleachedguide 3D-MASTERShade system, which utilizes a multiple color spectrophotometer andincludes half lightness levels. A difference of one shade is referred toherein as a “shade value unit” (SVU). Thus, for example, a difference oftwo shades is a 2 SVU difference.

“Bleaching agent” as used herein refers to the active ingredient, orcombination of ingredients, in a composition that causes the lighteningand/or removal of the chromagens that contribute to the dark shade of atooth. A composition comprising a bleaching agent in an efficaciousamount is a bleaching composition.

As used herein, an “efficacious amount” of a bleaching agent is intendedto mean any amount of a bleaching agent that will result in toothwhitening, as defined herein, using methods of assessment known to theskilled artisan, as discussed above of a tooth, with one or moretreatments.

A “mixed agent bleaching composition” as used herein refers to acomposition comprising two or more bleaching agents, wherein thebleaching agents together are present in an efficacious amount.

As used herein, “cytotoxic” refers to the property of causing lethaldamage to mammalian cell structure or function. A composition is deemed“substantially non-cytotoxic” or “not substantially cytotoxic” if thecomposition meets the United States Pharmacopeia (USP) biologicalreactivity limits of the Agar Diffusion Test of USP <87> “BiologicalReactivity, in vitro,” (approved protocol current in 2007) when theactive ingredient is present in an efficacious amount.

As used herein, “reduced cytotoxicity” is a relative term wherein thecytotoxicity of a first composition is compared to a referencecomposition and wherein if the cytotoxicity of the first composition isless than that of the reference composition, the first composition isdeemed to have reduced cytotoxicity.

As used herein, “comparable cytotoxicity” is a relative term wherein thecytotoxicity of a first composition is compared to a referencecomposition and wherein if the cytotoxicity of the first composition isabout the same as that of the reference composition, the firstcomposition and the reference composition are deemed to have comparablecytotoxicity.

As used herein, “comparable bleaching efficacy” refers to a similardegree of whitening achieved using a first and a second bleachingcomposition under the same treatment conditions. For instance,composition A and composition B are deemed to have comparable bleachingefficacy if, under the same treatment conditions (e.g., two-30 minutetreatments) a comparable change in shade value units (e.g, 3 SVUs) isachieved by both compositions. Efficacy of bleaching can be assessed byan method known in the art.

As used herein, “enhanced bleaching efficacy” is a relative term whereinthe whitening efficacy of a first composition comprising at leastchlorine dioxide as a bleaching agent is compared to a secondcomposition under the same treatment conditions, wherein the first andsecond compositions have comparable cytotoxicity and the secondcomposition does not comprise chlorine dioxide as a bleaching agent. Forinstance, composition A is deemed to provide enhanced bleaching efficacyrelative to composition B if, under the same treatment conditions (e.g.,two-30 minute treatments) composition A yields a greater improvement inshade value units than composition B.

As used herein, “irritating” refers to the property of causing a localinflammatory response, such as reddening, swelling, itching, burning, orblistering, by immediate, prolonged, or repeated contact. A compositionis deemed “substantially non-irritating” or “not substantiallyirritating” if the composition is judged to be slightly or notirritating using any standard method for assessing oral mucosalirritation. Non-limiting examples of such methods include: HET-CAM(hen's egg test-chorioallantoic membrane); slug mucosal irritation test;and in vitro tests using tissue-engineered oral mucosa.

As used herein, “reduced irritation” is a relative term wherein theirritation associated with a first composition is compared to theirritation associated with a reference composition and wherein if theirritation of the first composition is less than that of the referencecomposition, the first composition is deemed to have reduced irritation.

As used herein, “hard tooth tissue” refers to at least one of enamel anddentin.

As used herein, “hard tooth tissue damage” refers to at least one of areduction of microhardness of enamel, a reduction of microhardness ofdentin, an increase in the surface roughness of enamel and an increasein the surface roughness of dentin.

As used herein, “reduced hard tooth tissue damage” is a relative termwherein the hard tooth tissue damage associated with a first compositionis compared to the hard tooth tissue damage associated with a referencecomposition and wherein if the hard tooth tissue damage of the firstcomposition is less than that of the reference composition, the firstcomposition is deemed to have reduced hard tooth tissue damage.

As used herein, “soft tissue” refers generally to mucosal tissue. Softoral tissue includes buccal mucosa, and other oral cavity mucosa,e.g.,soft palate mucosa, floor of mouth mucosa and mucosa under the tongue,as well as the lips.

As used herein, a composition “does not substantially damage hard toothtissue” if one or more of the following is met for a tooth aftertreatment relative to the tooth prior to treatment: 1) enamelmicrohardness is decreased by an amount less than about 15% and/or thereduction is not statistically significant at the 5% confidence level;2) dentin microhardness is decreased by an amount less than about 15%and/or the reduction is not statistically significant at the 5%confidence level; 3) enamel surface roughness is increased by an amountno more than about 20% and/or the increase is not statisticallysignificant at the 5% confidence level; and 4) dentin surface roughnessis increased by an amount no more than about 8% and/or the increase isnot statistically significant at the 5% confidence level.

As used herein, “oxy-chlorine anion” refers to chlorite (ClO₂ ⁻) and/orchlorate (ClO₃ ⁻) anions.

As used herein, “substantially pure chlorine dioxide solution” refers toa solution of chlorine dioxide that has a non-cytotoxic concentration ofoxy-chlorine anion. As used herein, “substantially pure chlorine dioxidesolution” also refers to a concentrated solution of chlorine dioxidethat contains a concentration of oxy-chlorine anion that upon dilutionto an efficacious amount of chlorine dioxide is not cytotoxic withrespect to the concentration of oxy-chlorine anion.

The phrase “thickened fluid composition” encompasses compositions whichcan flow under applied shear stress and which have an apparent viscositywhen flowing that is greater than the viscosity of the correspondingaqueous chlorine dioxide solution of the same concentration. Thisencompasses the full spectrum of thickened fluid compositions,including: fluids that exhibit Newtonian flow (where the ratio of shearrate to shear stress is constant and viscosity is independent of shearstress), thixotropic fluids (which require a minimum yield stress to beovercome prior to flow, and which also exhibit shear thinning withsustained shear), pseudoplastic and plastic fluids (which require aminimum yield stress to be overcome prior to flow), dilantant fluidcompositions (which increase in apparent viscosity with increasing shearrate) and other materials which can flow under applied yield stress.

A “thickener component,” as the phrase is used herein, refers to acomponent that has the property of thickening a solution or mixture towhich it is added. A “thickener component” is used to make a “thickenedfluid composition” as described above.

The phrase “apparent viscosity” is defined as the ratio of shear stressto shear rate at any set of shear conditions which result in flow.Apparent viscosity is independent of shear stress for Newtonian fluidsand varies with shear rate for non-Newtonian fluid compositions.

The term “particulate” is used herein to refer to all solid materials.By way of a non-limiting example, particulates may be interspersed witheach other to contact one another in some way. These solid materialsinclude particles of any size, and combinations of particles ofdifferent sizes.

As used herein, “remineralization” refers to the process of repair ofacid damaged tooth structure by the recrystallization of mineral saltson or within the tooth architecture.

As used herein, “demineralization” refers to the process of mineral lossfrom teeth caused by acid, chelating agents or other accelerants ofdissolution. Demineralization can occur on tooth surfaces and/or belowtooth surfaces, depending on the composition of the demineralizingagent, the contacting medium, the concentration and the pH. As usedherein, a method is considered to have improved bleaching when theextent of whitening achieved by the method is greater by at least one(1) shade value unit than that achieved by a reference method under thesame treatment conditions regarding frequency of treatment and durationof treatment and the bleaching compositions used in the two methods haveat least one of comparable irritation, comparable cytotoxicity andcomparable hard tooth tissue damage, and wherein the reference methoddoes not use chlorine dioxide as a bleaching agent. Improved bleachingalso encompasses faster bleaching, wherein the same degree of whiteningis achieved by a method in a shorter total treatment time compared to areference method. Total treatment time is the sum of all bleachingtreatments administered to achieve the given degree of whitening.

As used herein, a method is considered to have reduced irritation whenthe extent of irritation associated with the method is less than thatobserved for a reference method under the same treatment conditionsregarding frequency of treatment and duration of treatment and thebleaching compositions used in the two methods have at least one ofcomparable bleaching efficacy, comparable cytotoxicity and comparablehard tooth tissue damage, and wherein the reference method does not usechlorine dioxide as a bleaching agent.

As used herein, a method is considered to have reduced hard tooth tissuedamage when the extent of hard tooth tissue damage associated with themethod is less than that observed for a reference method under the sametreatment conditions regarding frequency of treatment and duration oftreatment and the bleaching compositions used in the two methods have atleast one of comparable bleaching efficacy, comparable cytotoxicity andcomparable irritation, and wherein the reference method does not usechlorine dioxide as a bleaching agent.

Description

The compositions, kits and methods of use described herein spring inpart from the discovery that potent and rapid tooth whitening can beachieved using chlorine dioxide compositions that are non-cytotoxic,cause minimal damage to hard tooth tissues and are substantiallynon-irritating to soft tissues of the mouth. As shown herein,cytotoxicity of chlorine dioxide-containing compositions resultspredominantly from the presence of oxy-chlorine anions, and not from thepresence of chlorine, which can be a product of chlorine dioxidedecomposition. Further as shown herein, substantially non-cytotoxic,non-irritating and non-hard tissue damaging compositions comprisingchlorine dioxide as a bleaching agent provide rapid and extensive toothwhitening. Indeed, the rate and extent of tooth whitening achieved bysuch compositions was found to be superior to over-the-counterperoxide-based tooth whitening compositions and comparable to certainprofessional peroxide-based tooth whitening compositions. In someembodiments, the tooth surface can be whitened by at least about 3 shadevalue units, at least about 5 shade value units, or at least about 6shade value units. Advantageously, substantially non-cytotoxicchlorine-dioxide containing compositions do not adversely affect enamelor dentin microhardness to a significant extent. Furthermore, thecompositions do not substantially increase surface roughness of teeth,even after extended contact with the composition. Thus, tooth whiteningis obtained without substantially damaging hard tooth tissue. It isbelieved therefore that tooth whitening in accordance with the methodwill have reduced or substantially reduced tooth sensitivity and softtissue irritation, compared to peroxide-based tooth whitening productscurrently available. It is further contemplated that the extent and/orrate of rebound will be reduced, compared to peroxide-based toothwhitening products currently available.

Thus, a method of whitening a tooth is provided, the method comprisingcontacting a tooth surface with an efficacious amount of a composition,wherein the composition comprises chlorine dioxide as a bleaching agent.The composition is substantially non-cytotoxic, substantiallynon-irritating to soft tissues in the mouth and does not substantiallydamage hard tooth tissue.

The discovery that chlorine dioxide compositions can be made to besubstantially non-cytotoxic also leads to a mixed agent bleachingcomposition comprising chlorine dioxide and at least a second bleachingagent, wherein the mixed agent bleaching composition has enhancedbleaching efficacy at equivalent cytotoxicity to the second bleachingagent alone. The mixed agent bleaching composition and methods of usingit in a method of whitening a tooth are also provided.

I. Composition

The substantially non-cytotoxic composition used in the practice of themethod is an aqueous fluid that comprises chlorine dioxide, or thereactants for generating chlorine dioxide, as a bleaching agent. Inpreferred embodiments, the composition comprises a thickener componentwhich renders the composition a thickened aqueous fluid. In anotherpreferred embodiment, the composition is an oral rinse that may be heldin the mouth in contact with teeth, as well as soft tissue.

Compositions useful in the practice of the method comprise at leastabout 5 ppm chlorine dioxide, preferably at least about 20 ppm, and morepreferably at least about 30 ppm. Typically, the amount of chlorinedioxide can be up to about 1000 ppm, preferably up to about 700 ppm,more preferably up to about 500 ppm and more preferably still up toabout 200 ppm. In certain embodiments, the chlorine dioxideconcentration ranges from about 5 to about 700 ppm, preferably fromabout 20 to about 500 ppm, and most preferably from about 30 to about200 ppm chlorine dioxide. In one embodiment, the composition comprisesabout 30 to about 40 ppm chlorine dioxide. In one embodiment, thecomposition comprises about 30 ppm. In another embodiment, thecomposition comprises about 40 ppm.

Soft tissue irritation can result from highly reactive oxygen species,such as those found in peroxide based compositions. Soft tissueirritation can also result from extremes of pH, both acidic and basic.To minimize soft tissue irritation of the chlorine dioxide containingcomposition, the substantially non-cytotoxic composition has a pH of atleast 3.5. To minimize possible hard surface erosion, the compositionhas a pH of at least about 4.5. Preferably, the composition has a pH ofat least 5, and more preferably still, greater than about 6. In certainembodiments, the pH ranges from about 4.5 to about 11, more preferablyfrom about 5 to about 9, and more preferably still, greater than about 6and less than about 8. In one embodiment, the pH is about 6.5 to about7.5. Irritation is not believed to result from the concentration ofoxy-chlorine anions.

For compositions comprising chlorine dioxide, as shown herein,cytotoxicity results predominantly from the presence of oxy-chlorineanions. Accordingly, a composition comprising chlorine dioxide thatcomprises zero milligram (mg) oxy-chlorine anion per gram composition tono more than about 0.25 mg oxy-chlorine anion per gram composition,preferably zero to about 0.24, 0.23, 0.22, 0.21, or 0.20 mg oxy-chlorineanion per gram composition, more preferably zero to about 0.19, 0.18,0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, or 0.10 mg oxy-chlorine anionper gram composition and more preferably still from zero to about 0.09,0.08, 0.07, 0.06, 0.05 or 0.04 mg oxy-chlorine anion per gramcomposition, absent other constituents that contribute to cytotoxicity,is substantially non-cytotoxic.

A substantially non-cytotoxic composition comprising chlorine dioxidecan be prepared using a substantially pure chlorine dioxide solutionhaving a neutral pH. Preferably, the solution has a pH from about 5 toabout 9, and more preferably, from about 6.5 to about 7.5. One source ofa substantially pure chlorine dioxide solution is chlorine dioxide isprepared using an ASEPTROL (BASF Corp., Florham Park, N.J.) material,which are described in commonly-assigned U.S. Pat. Nos. 6,432,322 and6,699,404. These patents disclose solid bodies for preparing highlyconverted solutions of chlorine dioxide when added to water. The solidbody comprises a metal chlorite such as sodium chlorite, an acid sourcesuch as sodium bisulfate and optionally a source of free halogen such asthe sodium salt of dichloroisocyanuric acid or a hydrate thereof.ASEPTROL materials provide a way to efficiently generate chlorinedioxide at substantially neutral pH, thus avoiding tooth-compatibilityproblems existing with earlier, acidic chlorine dioxide based oralproducts. ASEPTROL material in an aqueous fluid has an extremely highconversion rate, resulting in high concentrations of chlorine dioxideand low concentrations of oxy-chlorine anion.

Another method of preparing substantially pure chlorine dioxide is toprepare a chlorine dioxide source solution by any known method, thenbubbling air through that solution (sparging) and into a secondcontainer of deionized water, to prepare the product solution ofsubstantially pure chlorine dioxide. Only ClO₂ and possibly some watervapor is transferred from the source solution to the product solution.All the salt ingredients remain behind in the source solution. Thus,there are no oxy-chlorine anions in the substantially pure productsolution. While the chlorine dioxide may undergo a degree ofdecomposition, the rate is relatively slow. By keeping the solutioncapped and protected from ultraviolet exposure, the decomposition ratecan be slowed to a rate of about 5 to about 25% reduction in chlorinedioxide in 7 days. Substantially pure chlorine dioxide may also beprepared using a pervaporation technique, such as that disclosed in U.S.Pat. No. 4,683,039. In addition, a metal chlorite and an acid source canbe reacted in solution to yield high conversion to chlorine dioxide andproduce a greater than 2000 ppm chlorine dioxide solution. Theconcentrated solution can then be buffered to a neutral pH. Similarly, achlorine dioxide solution can be prepared the composition described inU.S. Pat. No. 5,399,288, which yields a high concentration chlorinedioxide solution at acidic pH. The concentrated solution can then bebuffered to achieve a substantially neutral pH to prepare asubstantially pure chlorine dioxide solution.

Oxy-chlorine anions can be measured in these solutions using any methodknown to those skilled in the art, including ion chromatographyfollowing the general procedures of EPA test method 300 (Pfaff, 1993,“Method 300.0 Determination of Inorganic Anions by Ion Chromatography,”Rev. 2.1, US Environmental Protection Agency) or a titration methodbased on an amperometric method (Amperometric Method II in Eaton et al,ed., “Standard Methods for the Examination of Water and Wastewater”19^(th) edition, American Public Health Association, Washington D.C.,1995). Alternatively, oxy-chlorine anions may be measured by a titrationtechnique equivalent to the amperometric method, but which uses theoxidation of iodide to iodine and subsequent titration with sodiumthiosulfate to a starch endpoint in place of the amperometric titration;this method is referred to herein as “pH 7 buffered titration.” Achlorite analytical standard can be prepared from technical grade solidsodium chlorite, which is generally assumed to comprise about 80% byweight of pure sodium chlorite.

To prepare a thickened aqueous composition comprising chlorine dioxidethat is substantially not cytotoxic, non-irritating and does not damagehard tooth tissue, the substantially pure chlorine dioxide solution canthen be combined with a thickener component and an aqueous medium. Thematerials and methods also encompass a two-component whitening systemcomprising a first component comprising a substantially pure chlorinedioxide solution and a second component comprising a thickener componentin an aqueous medium. Combination of the first and second componentsyields a non-cytotoxic composition comprising an amount of chlorinedioxide efficacious for tooth whitening. Chlorine dioxide in solutionwill decompose over time. To avoid problems arising from suchdecomposition, including loss of efficacy and generation of chloriteanions, the substantially pure chlorine dioxide solution is preparedimmediately before its combination with a thickener component and anaqueous medium. In addition, the composition is prepared immediatelybefore its use.

“Immediately before” as used herein refers to a period no greater thanthat which would result in diminished efficacy or evidence ofcytotoxicity. Generally, “immediately before” is less than about 14days, and preferably no greater than about 24 hours and more preferablyno greater than about 2 hours. Preferably, the substantially purechlorine dioxide solution is prepared within about 8 hours of thepreparation of the composition. Precautions are also taken to avoidexposing the chlorine dioxide solution or the prepared composition tostrong ultraviolet light or elevated temperature (e.g., temperaturegreater than ambient temperature, about 25° C.).

Methods of preparing substantially non-cytotoxic thickened compositionscomprising chlorine dioxide are also disclosed in commonly-assigned U.S.provisional patent application No. 61/150,685, filed Feb. 6, 2009,entitled “Non-Cytotoxic Chlorine Dioxide Fluids,” incorporated herein byreference in its entirety.

Methods of preparing thickened compositions comprising chlorine dioxideare also disclosed in commonly-assigned US Pat. Publication Nos.2006/0169949 and 2007/0172412, which are herein incorporated byreference in their entirety. In practicing the methods described inthese two publications, steps must be taken (as described herein) tocontrol the oxy-chlorine concentration so as to produce a non-cytotoxiccomposition.

A substantially non-cytotoxic composition comprising chlorine dioxidecan also be prepared using a particulate precursor of ClO₂ and anaqueous thickened fluid composition. Another aspect encompasses atwo-component whitening system comprising a first component comprising aparticulate precursor of chlorine dioxide and a second componentcomprising a thickener component in an aqueous medium. Combination ofthe first and second components yields a non-cytotoxic compositioncomprising an amount of chlorine dioxide efficacious for toothwhitening. In an embodiment, the components of the system are adapted tobe admixed and applied to the teeth from a dental tray for sustainedcontact. Precursors of ClO₂ include metal chlorites, metal chlorates, anacid source and an optional halogen source. The particulate precursormay comprise one of these or any combination of these. Preferably theparticulate precursor is an ASEPTROL product, more preferably it isASEPTROL S-Tab2. ASEPTROL S-Tab2 has the following chemical compositionby weight (%): NaClO₂ (7%); NaHSO₄ (12%); NaDCC (1%); NaCl (40%); MgCl₂(40%). Example 4 of U.S. Pat. No. 6,432,322 describes an exemplarymanufacture process of S-Tab2. Granules are produced, either bycomminuting pressed S-Tab2 tablets, or by dry roller compaction of thenon-pressed powder of the S-Tab2 components, followed by breakup of theresultant compacted ribbon or briquettes, and then screening to obtainthe desired size granule. Upon exposure to water or an aqueous thickenedfluid, chlorine dioxide is generated from the ASEPTROL granules. In oneembodiment, a substantially non-cytotoxic composition comprisingchlorine dioxide is prepared by combining −40 mesh granules with anaqueous thickened fluid. In one aspect, the thickener component of thethickened fluid is carboxymethylcellulose. Preferably, the aqueousthickened fluid is prepared sufficiently in advance of combining withthe ASEPTROL granules to enable the complete hydration of the thickenercomponent. In one embodiment, the thickened fluid composition is formedby adding high viscosity NaCMC powder to distilled water. The NaCMC isallowed to hydrate for at least 8 hours, and then the mixture is stirredto homogenize it. A substantially non-cytotoxic composition for toothwhitening is then prepared by mixing the sized ASEPTROL granules withthe NaCMC thickened fluid composition.

The thickened fluid composition may also be formed in situ, whereinsaliva serves as the aqueous medium. In one embodiment, a mixture ofASEPTROL granules and a thickener component is formed into a shape, forinstance by addition of a malleable wax, and the shape is then appliedto teeth. Saliva activates the granules, forming chlorine dioxide andthe thickener component hydrates, thereby forming the thickened fluidcomposition in situ. In another embodiment, a mixture of ASEPTROLgranules and a thickener component is placed on a dental strip or adental film or in a dental tray. A dental strip refers to asubstantially planar object made of a plastic backbone that issufficiently flexible to affix to teeth. A dental film refers to asubstantially planar object made of a pliable, conformable material thatcan be substantially fitted to the surface of teeth. Optionally, thedental strip is dissolvable in an aqueous medium, such as saliva. Thestrip, film or tray is positioned on teeth, and saliva serves as theaqueous medium as described above to produce the thickened fluidcomposition in situ. Alternatively, the mixture on the strip or tray iscontacted with water or aqueous medium prior to positioning on theteeth.

There is no extremely accurate method for measuring oxy-chlorine aniondirectly in a thickened fluid composition. This value can be accuratelyestimated, however, by measuring the oxy-chlorine anion in the aqueoussolution (prior to thickening), and adjusting the final concentration onthe basis of weight of the final thickened fluid. The titration methoddescribed elsewhere herein is contemplated as useful in assessing boththe chlorine dioxide concentration and the oxy-chlorine anionconcentration in thickened fluid compositions. It is contemplated thatoxy-chlorine anions in a thickened fluid composition can be measuredusing ion chromatography as described elsewhere herein, provided stepsare taken to preclude fouling of the column by the hydrated thickenercomponent. One such step is the use of molecular weight filters toremove the hydrated thickener component, such as hydrated CMC, prior toapplication to the chromatography column. If necessary, the thickenedfluid composition may be diluted with water, prior to analysis, toreduce its viscosity or otherwise allow it to be more readily tested.One of skill in the art can readily determine empirically whether agiven formulation has a sufficiently low oxy-chlorine concentration bydetermining if the formulation is cytotoxic using USP biologicalreactivity limits of the Agar Diffusion Test of USP <87>.

The aqueous thickened fluid composition used in practicing the methodmay comprise any thickener component in an aqueous medium, wherein thethickened fluid composition is non-cytotoxic and non-irritating to softtissues, in particular oral mucosa, and causes minimal damage to hardtissues, such as tooth enamel and dentin. In addition, the thickener ispreferably not adversely affected by the bleaching agent on the timescale of composition preparation and use in treatment. Many thickeneragents are known in the art, including, but not limited to carbomers(e.g., CARBOPOL thickeners, Lubrizol Corp., Wickliffe, Ohio),carboxymethylcellulose (CMC), ethylcellulose, hydroxyethylcellulose,hydroxypropyl cellulose, natural smectite clays (e.g., VEEGEM, R.T.Vanderbilt Co., Norwalk, Conn.), synthetic clays (e.g., LAPONITE(Southern Clay Products, Gonzales, Tex.), methylcellulose,superabsorbent polymers such as polyacrylates (e.g., LUQUASORB 1010,BASF, Florham Park, N.J.), poloxamers (PLURONIC, BASF, Florham Park,N.J.), polyvinyl alcohol, sodium alginate, tragacanth, and xanthan gum.Such thickening agents may be categorized into four groups: naturalhydrocolloids (also referred to as “gum”), semisynthetic hydrocolloids,synthetic hydrocolloids, and clay. Some examples of naturalhydrocolloids include acacia, tragacanth, alginic acid, carrageenan,locust bean gum, guar gum, and gelatin. Non-limiting examples ofsemisynthetic hydrocolloids include methylcellulose and sodiumcarboxymethylcellulose. Some examples of synthetic hydrocolloids (alsoreferred to as “polymers” including polymers, cross-linked polymers, andcopolymers) include polyacrylates, superabsorbent polymers, highmolecular weight polyethylene glycols and polypropylene glycols,polyethylene oxides and CARBOPOL. Non-limiting examples of clay(including swelling clay) include LAPONITE, attapulgite, bentonite andVEEGUM. Preferably the thickener component is a semisynthetichydrocolloid. More preferably, the thickener component is a highviscosity sodium carboxymethylcellulose (NaCMC powder).

CMC is a cellulose derivative with carboxymethyl groups (—CH₂—COOH)bound to some of the hydroxyl groups of the glucopyranose monomers thatmake up the cellulose backbone. It is synthesized by thealkali-catalyzed reaction of cellulose with chloroacetic acid. The polar(organic acid) carboxyl groups render the cellulose soluble andchemically reactive. The functional properties of CMC depend on thedegree of substitution of the cellulose structure (i.e., how many of thehydroxyl groups have taken part in the substitution reaction), and chainlength of the cellulose backbone structure.

CMC is available in a range of viscosity grades and to USP standards.High viscosity CMC, such as type CA194 from Spectrum ChemicalManufacturing Company, has a viscosity of between 1500 and 3000 cps at25° C. at 1% concentration in water.

The use of a bleaching composition comprising a mixture of bleachingagents is also contemplated, specifically chlorine dioxide as a firstbleaching agent and at least a second bleaching agent. Exemplary secondbleaching agents include alkali metal percarbonates (such as sodiumpercarbonate), carbamide peroxide, sodium perborate, potassiumpersulfate, calcium peroxide, zinc peroxide, magnesium peroxide,hydrogen peroxide complexes (such as a PVP-hydrogen peroxide complex),hydrogen peroxide, and combinations thereof. In some embodiments, thesecond bleaching agent is a peroxide-based agent. Peroxide-basedbleaching agents can be both irritating and cytotoxic at efficaciousoxidizing concentrations. It is contemplated that a substantiallynon-cytotoxic chlorine dioxide composition combined with one or morecytotoxic bleaching agents will yield a potent mixed agent bleachingcomposition that is associated with a reduced irritation, and possiblyalso reduced cytotoxicity, relative to a composition comprising asufficient quantity of the bleaching agent to have comparable bleachingefficacy in the absence of chlorine dioxide. Similarly, it iscontemplated that a substantially non-cytotoxic chlorine dioxidecomposition combined with one or more cytotoxic bleaching agents willyield a mixed agent bleaching composition having comparable cytotoxicityand enhanced bleaching efficacy relative to a reference compositioncomprising the one or more cytotoxic bleaching agents in the sameamounts as in the mixed agent composition and not comprising chlorinedioxide.

It is also contemplated that a mixed agent bleaching composition will beassociated with a reduced hard tooth tissue damage relative to acomposition of comparable bleaching efficacy in the absence of chlorinedioxide. Specifically, it is expected that the adverse changes in enamelmicrohardness and dentin microhardness observed for a composition ofabout 36% hydrogen peroxide will be of a lesser amount using a mixedagent bleaching composition having comparable bleaching efficacy as theabout 36% hydrogen peroxide composition.

Additionally, it is contemplated that a mixed agent bleachingcomposition will exhibit a synergistic bleaching efficacy relative tothe bleaching efficacies of the individual bleaching agents. In otherwords, the tooth whitening efficacy of the mixed bleaching agent isgreater than the sum of the efficacies of the individual bleachingagents, at the same concentrations and administered under the sameconditions. Synergy can be advantageous as it may enable a desiredwhitening endpoint to be reached using a more rapid treatment scheduleor employing a reduced concentration of one or all of the bleachingagents. In preferred embodiments, the concentration of the one or morenon-chlorine dioxide bleaching agent is reduced. This reduction isexpected to lead to further reductions in irritation, hard tooth surfacedamage and possible cytotoxicity of the composition, relative to acomposition with a higher concentration of the one or morenon-chlorine-dioxide bleaching agents.

The mixed agent bleaching composition can be prepared by the methodsdescribed herein for a non-cytotoxic composition, by incorporating atleast one other bleaching agent. The mixed agent bleaching compositionmay be a fluid or a thickened fluid. The precursor composition usefulfor preparing the substantially non-cytotoxic thickened fluidcomposition, as described in commonly-assigned application entitled“Non-Cytotoxic Chlorine Dioxide Fluids” may also be used to prepare anoxidizing composition of reduced cytotoxicity by addition of at leastone other oxidizing agent to the prepared non-cytotoxic thickened fluidcomposition. A mixed agent precursor composition comprising aparticulate precursor of chlorine dioxide and a particulate precursor ofat least a second bleaching agent is contemplated. For instance, a mixedagent precursor composition comprising a particulate precursor ofchlorine dioxide and one or more of sodium perborate, potassiumpersulfate, carbamide peroxide, or an alkali metal percarbonate, whencontacted with an aqueous fluid, will generate both chlorine dioxide andhydrogen peroxide. The particulate matter is preferably anhydrous orotherwise stabilized to preclude premature activation. Methods tostabilize components are discussed, for instance, in commonly-assignedU.S. provisional patent application No. 61/150,685, filed Feb. 6, 2009,entitled “Non-Cytotoxic Chlorine Dioxide Fluids” and in US PatentPublication No. 2007/0172412, which are incorporated herein by referencein its entirety.

In some embodiments, the mixed agent bleaching composition compriseschlorine dioxide and a peroxide agent. Representative peroxide agentsinclude, but are not limited to, hydrogen peroxide, calcium peroxide,magnesium peroxide, zinc peroxide and carbamide peroxide. In someembodiments, the peroxide agent is hydrogen peroxide. The peroxide agentis present in the composition at greater than about 1% (by weight) andless than about 36%, more preferably, less than about 30%, less thanabout 10%, and more preferably still, about 6% or less. A mixed agentbleaching composition may further comprise other components, such asthose described elsewhere herein. The optional components are preferablyrelatively resistant to oxidation by the bleaching agents.

To minimize irritation due to extreme pH, the mixed agent compositionspreferably have a pH greater than 3.5, more preferably greater thanabout 5 and more preferably still, greater than about 6. As describedelsewhere herein, in certain embodiments, the pH ranges from about 4.5to about 11, more preferably from about 5 to about 9, and morepreferably still, greater than about 6 and less than about 8. It isknown that low pH can also cause hard surface damage, specifically acidetching and/or tooth decalcification. Thus, in one embodiment, the mixedagent bleaching composition has a pH of less than or equal to about 5and further comprises a remineralizing agent to prevent or repairmineral loss from the enamel that may result from thenon-chlorine-dioxide bleaching agent. Exemplary remineralizing agentsinclude a source of calcium ions, a source of fluoride ions, a source ofphosphate ions and the like. A single remineralizing agent may be added,or a combination of agents. Optionally, the mixed agent bleachingcomposition having pH less than or equal to about 5 further comprises adesensitizing agent, such as, but not limited to, potassium nitrate,other potassium salts, citric acid, citrates, and sodium fluoride.Remineralizing agents and desensitizing agents are well known in theart.

Further provided is a three-component whitening system comprising afirst component comprising a substantially pure chlorine dioxidesolution as a first bleaching agent, a second component comprising athickener component in an aqueous medium and a third componentcomprising a second bleaching agent. Exemplary agents for the secondbleaching agent are discussed elsewhere herein. In one embodiment, thethree-component whitening system is used to prepare a bleachingcomposition comprising a mixture of bleaching agents. In anotherembodiment, the three-component whitening system is used to prepare afirst bleaching composition by combining the first and second componentsto yield a non-cytotoxic chlorine dioxide composition. The thirdcomponent may be in the form of a second bleaching composition.Alternatively, the third component is combined with a fourth componentcomprising a thickener component in an aqueous medium to prepare thesecond bleaching composition. The teachings regarding the two-componentsystem are applicable to the first and second components of the threecomponent system. For instance, the first component may comprise aparticulate precursor of chlorine dioxide and the second component maycomprise a thickener component in an aqueous medium. The two bleachingcompositions may be used in a method of whitening teeth comprisingsequential and alternating treatment with the first bleachingcomposition and the second bleaching composition.

The compositions useful in the practice of the method are fluids. Insome embodiments, the fluid is a thickened fluid having flow propertiessuitable for applying the fluid to a tooth surface and leaving the fluidin place for the duration of a tooth whitening treatment (e.g., about 5to about 60 minutes). Accordingly, a pseudoplastic composition with asufficient yield point to retains its shape when applied to teeth butlow enough to be readily removed by wiping is advantageous in practicingthe method. In embodiments where the composition is contacted to a toothusing a dental tray, strip or similar device, the composition shouldhave sufficient adhesion to hold the device in place. Exemplary adhesionagents are disclosed in US Pat. Publication No. 2008/0025925.

The compositions used in the method may optionally comprise othercomponents. Such components include, but are not limited to, sweeteners,flavorants, coloring agents and fragrances. Sweeteners include sugaralcohols. Exemplary sugar alcohols include sorbital, xylitol, lactitol,mannitol, maltilol, hydrogenated starch hydrolysate, erythritol,reducing paratinose and mixtures thereof. Flavoring agents include,e.g., natural or synthetic essential oils, as well as various flavoringaldehydes, esters, alcohols, and other materials. Examples of essentialoils include oils of spearmint, peppermint, wintergreen, sassafras,clove, sage, eucalyptus, marjoram, cinnamon, lemon, lime, grapefruit,and orange. Coloring agents include a colorant approved forincorporation into a food, drug or cosmetic by a regulatory agency, suchas, for example, FD&C or D&C pigments and dyes approved by the FDA foruse in the United States. Fragrances include menthol, menthyl acetate,menthyl lactate, camphor, eucalyptus oil, eucalyptol, anethole, eugenol,cassia, oxanone, α-irisone, propenyl guaiethol, thymol, linalool,benzaldehyde, cinnamaldehyde, N-ethyl-p-menthan-3-carboxamine,N,2,3-trimethyl-2-isopropylbutanamide, 3-(1-methoxy)-propane-1,2-diol,cinnamaldehyde glycerol acetal (CGA), menthone glycerol acetal (MGA) andthe like.

Other optional components for the composition include: antibacterialagents (in addition to chlorine dioxide), enzymes, malodor controllingagents (in addition to chlorine dioxide), cleaning agents, such asphosphates, antigingivitis agents, antiplaque agents, antitartar agents,anticaries agents, such as a source of fluoride ion, antiperiodontitisagents, nutrients, antioxidants, and the like. Advantageously, themethod is expected to reduce or eliminate the need for agents to relievetooth sensitivity and agents for remineralizing the enamel. However,such agents may be included in the composition in some embodiments.Exemplary agents are well-known in the art. See, for instance, US Pat.Publication Nos. 2005/0287084; 2006/0263306; 2007/0259011; and2008/0044363.

It is preferred that all optional components are relatively resistant tooxidation by chlorine dioxide, since oxidation of composition componentsby chlorine dioxide will reduce the available chlorine dioxide foroxidation of chromagens on a tooth. In compositions comprising onlychlorine dioxide as a bleaching agent, “relatively resistant” means thatin the time scale of preparing and using the chlorine dioxide-containingcomposition the function of the optional component is not diminished,and the composition retains tooth whitening efficacy and remainssubstantially non-cytotoxic, substantially non-irritating, and does notsubstantially damage hard tooth tissue. In mixed agent bleachingcompositions comprising chlorine dioxide as a first bleaching agent,“relatively resistant” means that in the time scale of preparing andusing the mixed agent composition in a method, the function of theoptional component is not diminished, and the mixed bleaching agentcomposition retains tooth whitening efficacy and results in acceptablylow cytotoxicity, irritation, and hard tissue effects.

II. Method of Use

In one embodiment, the method is practiced by contacting a tooth surfacewith a substantially non-cytotoxic composition comprising chlorinedioxide as the bleaching agent in an efficacious amount. Advantageously,no pre-treatment of the gums is necessary in this embodiment, becausethe composition is substantially non-irritating and substantiallynon-cytotoxic. Advantageously, even after prolonged contact, thenon-cytotoxic composition does not substantially damage hard toothtissue.

In another embodiment, the method is practiced by contacting a toothsurface with a mixed agent bleaching composition in an efficaciousamount. In a preferred embodiment, the mixed agent bleaching compositionhas enhanced bleaching efficacy relative to a reference compositionhaving comparable cytotoxicity and comprising no chlorine dioxide, asdescribed elsewhere herein.

In yet another embodiment, the method is practiced by contacting a toothsurface with a first bleaching composition in an efficacious amount andthen contacting the tooth surface with a second bleaching composition inan efficacious amount. The first bleaching composition is asubstantially non-cytotoxic chlorine dioxide composition. The secondbleaching composition comprises a second bleaching agent. In anembodiment, the first step of the method comprises treatment with thefirst bleaching composition. The second step comprises treatment withthe second bleaching composition. For instance, the first step is atreatment with 200 ppm chlorine dioxide for 15 minutes and the secondstep is a treatment with 6% hydrogen peroxide for 15 minutes. In anotherembodiment, the first step of the method comprises treatment with thesecond bleaching agent, and the second step comprises treatment with thefirst bleaching agent. There can be one or more treatments with eachbleaching composition. Therefore, the method may comprise iterations ofthese two steps. The method may comprise steps alternating between thetwo bleaching compositions or may comprise two or more sequential stepsof treatment with one of the bleaching compositions, followed by atleast one step of treatment with the other bleaching composition. Thenumber and/and duration of treatments with the first bleachingcomposition may be the same or different as the number and/or durationof treatments with the second bleaching composition. The first bleachingcomposition may be identical in the plural steps or may be different,such as a different concentration of chlorine dioxide. Similarly, thesecond bleaching composition may be identical in the plural steps or maybe different, such as a different concentration of the bleaching agentand/or a different bleaching agent. Likewise, the duration of treatmentsteps may be the same or different for the first bleaching compositionand for the second bleaching composition. For instance, the first stepis a treatment for 10 minutes with a 10% hydrogen peroxide bleachingcomposition and a second step is a treatment for 20 minutes with a 35%hydrogen peroxide bleaching composition. Another non-limiting example isa first step being a treatment with 200 ppm chlorine dioxide bleachingcomposition for 15 minutes and a second step is a treatment with asecond bleaching composition comprising both a peroxide agent andchlorine dioxide. Other variations will be readily evident to theskilled artisan.

The method of whitening teeth comprising alternating treatment with afirst bleaching composition comprising chlorine dioxide as anon-cytotoxic composition and a second bleaching composition is expectedto achieve the same benefits of treatment with a bleaching compositioncomprising a mixture of chlorine dioxide and a second bleaching agent.Specifically, it is envisioned that the method will have one or more ofimproved bleaching, reduced irritation and reduced hard tooth tissuedamage. As envisioned for methods practiced using the mixed agentbleaching composition, it is also envisioned that the alternating stepsmethod will achieve synergistic bleaching efficacy that will enable thesame extent of tooth whitening to be achieved with fewer treatmentsteps, compared to a method using a single or plurality of non-chlorinedioxide bleaching agents.

The duration of contact with the tooth to achieve a measurable degree oftooth whitening can be readily determined by the skilled artisan in viewof the teachings herein. Generally, duration of contact ranges fromseconds to minutes, preferably at least about 60 seconds, morepreferably at least about 1, 2, 3, 4 or 5 minutes, still more preferablyabout 6, 7, 8, 9 or 10 minutes, yet more preferably about 11, 12, 13, 14or 15 minutes, though contact can range up to 16, 17, 18 19 or 20minutes, and further up to 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30minutes, and further up to 35, 40, 45, 50, 55, or 60 minutes or longerin some circumstances. In certain embodiments, duration of contactranges between about 1 and about 60 minutes, more preferably, from about5 minutes to about 30 minutes, and more preferably still, about 10 toabout 20 minutes. In a preferred embodiment, duration of contact for atreatment is about 15 minutes. Treatment frequency is also readilydetermined by skilled artisan armed with the present disclosure.Treatment may comprise one episode of tooth contact or more than oneepisode. Treatment episodes may be contiguous, separated in time (e.g.,a few hours to a few days, a few days to a few weeks, and also longerintervals including several months to a year or more) or both.

Contact between the composition and the tooth surface can be achieved byany of a number of well-known methods in the art. The composition can bebrushed or spread onto the tooth surface. The composition can be presenton a flexible strip or patch that can be pressed against and molded tothe tooth surface. The composition can be used as an oral rinse;accordingly, the composition can be held in the mouth and allowed tocontact the teeth either statically, or with agitation within the mouthusing, for example, the tongue and cheeks. The composition can be placedin a dental tray, which is then placed in contact with the teeth. Suchtrays may be custom made or non-custom made. Numerous devices useful inpracticing the method are disclosed in the art including, but notlimited to, U.S. Pat. Nos. 5,879,691; 6,551,579; 6,682,721;,6,848,905;6,896,518; 6,964,571; 7,004,756; 7,040,897 and US Pat. Publication Nos.2006/0223033; 2007/0298380; and 2008/0025925.

In some embodiments, the method is practiced using a dental tray.Preferably, the tray is custom made. Methods of making custom-made traysare well known in the art;

see, for instance, U.S. Pat. Nos. 6,106,284 and 6,425,759, and US Pat.Publication Nos. 2006/0183080 and 2008/0041400. In brief, an impressiontray is filled with an impression material, such as alginate. Theimpression tray is then positioned into the mouth of the patient so asto create a negative impression of the teeth in the impression material.After the negative impression has been formed, the negative impressioncreated in the impression tray is filled with a soft casting material,such as dental stone, plaster or epoxy. The impression tray is theninverted and mounted upon a pre-formed mounting device, such as a dentalcast tray or base. After the casting material has had an opportunity toharden, the impression tray is removed so that the casting materialforms a positive dental impression on the mounting surface. Anothermethod of preparing a custom dental tray makes use of a “boil and bite”material, which is made out of a thermoformable plastic such as ethylvinyl acetate (“EVA”) or polyethylene. A customized tray is created byheating the thermoformable plastic in boiling water causing it to meltat a biologically acceptable temperature, and then placing it directlyover an individual's teeth where it cools and retains its new shape. Topractice the method, the substantially non-cytotoxic compositioncomprising a bleaching agent is placed into the dental tray. The tray isthen positioned in the patient's mouth for the treatment episode.

It is also contemplated that administration of a chlorine dioxidecomposition may be made substantially non-cytotoxic by minimizing orprecluding contact of soft tissues with oxy-chlorine anions present inthe composition. Accordingly, as an example, devices comprising amicroporous barrier permeable to chlorine dioxide and substantiallynon-permeable to oxy-chlorine anions are envisioned. The chlorinedioxide composition may be completely or partially enclosed by such aselectively-permeable barrier. In some embodiments, the membrane ishydrophobic; the hydrophobic nature of the membrane prevents both anaqueous reaction medium and an aqueous recipient medium from passingthrough the membrane. Features to consider for the materials used forsuch a barrier include: hydrophobicity of the microporous material, poresize, thickness, and chemical stability towards the attack of chlorinedioxide, chlorine, chlorite, chlorate, chloride, acid and base. Ofcourse, for contact with soft tissues, the microporous barrier should besubstantially non-irritating and substantially non-cytotoxic,particularly in the time scale of typical use of the device. It isenvisioned that the chlorine dioxide composition utilized in such adevice need not be a thickened fluid, provided the device can be affixedto the tooth surface and enable the chlorine dioxide that permeatesthrough the membrane to contact the tooth surface.

Materials useful as such barriers are known in the art and includeexpanded polytetrafluoroethylene (e.g., GORE-TEX) andpolyvinylidenefluoride (PVDF). See, for instance, U.S. Pat. No.4,683,039. The procedure for formation of a expandedp6lytetrafluoroethylene is described in U.S. Pat. No. 3,953,566. Otherbarriers include micro-perforated polyethylene and polypropylene. Thematerial may be provided as a composite with supporting materials toprovide the structural strength required for use.

The pore sizes in the barrier may vary widely, depending on the desiredflow rate of the chlorine dioxide through the barrier. The pores shouldnot be so small as to prevent chlorine dioxide gas flow there throughbut also should not be so large that liquid flow is permitted.

The porosity of the barrier may vary widely, also depending upon thedesired flow rate of chlorine dioxide through the barrier.Considerations of barrier strength also dictate the porosity chosen.Generally, the barrier porosity varies from about 5% to about 98%.

The barrier may also comprise material which is permeable to chlorinedioxide in the absence of porosity. An example of such a material ispolystyrene film. Said barrier can deliver sufficient chlorine dioxidefor bleaching with reduced porosity relative to a barrier comprised ofmaterials which are substantially non-permeable to chlorine dioxide.Such films may, in fact, be non-porous.

Also contemplated is the use of reactants for the formation of ClO₂embedded in a polymeric material that is permeable to ClO₂ butsubstantially non-permeable to oxy-chlorine anions. See, for instance,U.S. Pat. No. 7,273,567.

As shown herein, a substantially non-cytotoxic composition comprisingchlorine dioxide as a bleaching agent has unexpected robust whiteningcapacity, which is a unique combination. Furthermore, a substantiallynon-cytotoxic composition comprising chlorine dioxide causes minimaldamage to hard surfaces, such as enamel and dentin, even during extendedcontact with an efficacious amount on a tooth surface. In someembodiments, microhardness of enamel contacted by a substantiallynon-cytotoxic composition is decreased less than about 15%, andpreferably less than about 10%, less than about 8% and more preferablystill, less than about 5%, relative to the enamel prior to contact. Insome embodiments, enamel microhardness is decreased less than about 1%after a total treatment time of about seven (7) hours, relative to theenamel prior to contact. In some embodiments, microhardness of dentincontacted by a substantially non-cytotoxic composition is decreased isdecreased less than about 15%, and preferably less than about 10%, andmore preferably still, less than about 8%, relative to the enamel priorto contact. In some embodiments, dentin microhardness is decreased lessthan about 8% after a total treatment time of about seven (7) hours,relative to the dentin prior to contact. Enamel and dentin surfaceroughness are also not substantially increased by contact with asubstantially non-cytotoxic composition. Preferably, surface roughnessis increased by no more than about 20%, more preferably no more thanabout 15%, more preferably no more than about 10%, and more preferablystill, no more than about 8%, relative to the surface roughness prior tocontact.

Thus, as shown herein, a substantially non-cytotoxic chlorinedioxide-comprising composition advantageously provides a high degree oftooth whitening rapidly, with substantially no damage to hard toothtissue, no irritation of soft mucosal tissue, and no tooth sensitivity.This is in marked contrast to currently-available products based onhydrogen peroxide, which are cytotoxic and an irritant to soft tissuesand frequently cause tooth sensitivity and demineralization. Forinstance, professional tooth lightening hydrogen peroxide productsrequire steps prior to treatment to protect the gums during treatment.Both over-the-counter and professional hydrogen peroxide-based productsoften cause undesirable side effects, including pain, sensitivity andirritation of soft and/or hard tissues. Additionally, the use of anon-cytotoxic chlorine dioxide-comprising composition has minimaladverse effect on enamel and dentin microhardness and surface roughness.This combination of highly effective tooth lightening coupled withminimal unpleasant side effects is very desirable, and not achieved inprior art efforts. The method does not require the use of an externalactivator, such as a bleaching light or laser, and tooth whitening equalor superior to current over-the-counter bleaching materials may beobtained in less overall treatment time. In addition, tooth whiteningequal or superior to current professional bleaching materials isobtained without cytotoxicity and irritation of soft tissue. Inparticular, the absence of soft tissue irritation advantageously permitsthe dental professional to proceed without a gum-protection step, whichcan take as long as about 90 minutes. Indeed, substantial contact withsoft oral tissue is possible without irritation or cytotoxicity.“Substantial contact with soft oral tissue” as used herein refers tocontact that is more than contact with gum tissue proximal to a treatedtooth. Thus, substantial contact includes, but is not limited to,contact with gum mucosa, cheek mucosa and tongue mucosa tissue.Furthermore, the minimal effect on enamel and dentin microhardnessreduces or eliminates the need for tooth sensitivity treatment and/or aremineralization procedure, both of which are often required withhydrogen peroxide-based tooth lightening products. In addition, it hasbeen suggested that the increased surface roughness observed afterhydrogen peroxide bleaching provides an increased surface, which mayfacilitate early restaining of the teeth after whitening treatment. Themethod advantageously does not increase surface roughness. Accordingly,it is contemplated that the method will also provide a reduced rateand/or extent of rebound after whitening.

Chlorine dioxide slowly decays over time. Thus, when the method ispracticed with a composition comprising chlorine dioxide, to maximizethe tooth whitening potency of the composition and to assurenon-cytotoxicity, the composition is prepared immediately before use oris prepared in situ, as described elsewhere herein. Preparation can beaccomplished by methods described in commonly-assigned U.S. provisionalapplication No. 61/150,685, filed Feb. 6, 2009, entitled “Non-CytotoxicChlorine Dioxide Fluids.”

In a preferred embodiment, a particulate precursor of chlorine dioxideis present in a first dispenser, such as a syringe, and a thickenercomponent in an aqueous medium is present in a second dispenser. Theaqueous thickened fluid in the second dispenser can be added directly tothe particulate mixture in the first dispenser, the combination allowedto react to produce ClO₂, and then mixed until homogeneous.Alternatively, an aqueous medium can be added to the particulateprecursor to prepare a substantially pure chlorine dioxide solution. Theappropriate amount of this solution is then mixed with the aqueousthickener in the other dispenser. In this embodiment, the aqueousmedium, the particulate precursor and the thickener component can be inseparate dispensers, such as syringes. The aqueous medium is thedispensed into the particulate precursor to prepare a substantially purechlorine dioxide. Both these embodiments are advantageously practicedusing syringes as the dispenser. In either embodiment, two syringes canbe connected to each other as appropriate, and the contents combined bydispensing the contents of one syringe into the other, then dispensingthe mixture back into the other syringe until the mixture ishomogeneous. In another embodiment, the dispensers are the barrels of amultiple barrel syringe, such as a dual barrel syringe. Other devices toprepare and dispense the composition are described in commonly-assigned“Non-Cytotoxic Chlorine Dioxide Fluids.”

III. Kits and Other Articles of Manufacture

Also provided herein a kit comprising the composition, or theingredients therefore, and an instructional material, which describesusing the composition in a method of whitening a tooth. As used herein,an “instructional material,” includes a publication, a recording, adiagram, or any other medium of expression which can be used tocommunicate the usefulness of the composition and/or compound in a kit.The instructional material of the kit may, for example, be affixed to acontainer that contains the compound and/or composition or be shippedtogether with a container which contains the compound and/orcomposition. Alternatively, the instructional material may be shippedseparately from the container with the intention that the recipient usesthe instructional material and the compound cooperatively. Delivery ofthe instructional material may be, for example, by physical delivery ofthe publication or other medium of expression communicating theusefulness of the kit, or may alternatively be achieved by electronictransmissions for example by means of a computer, such as by electronicmail, or download from a website.

In an embodiment, the kit comprises two dispensers useful for preparingthe composition. One dispenser comprises a particulate precursor ofchlorine dioxide. The second dispenser comprises a thickener componentin an aqueous medium.

In another embodiment, the kit comprises a two-compartment container.One compartment comprises a particulate precursor of chlorine dioxide.The second compartment comprises a thickener component in an aqueousmedium. Optionally, the container comprises a third compartment forcombining some or all of the contents of the two other compartments.

In some embodiments of the kit, the particulate precursor is ASEPTROLgranules, preferably ASEPTROL S-Tab2 granules. In some embodiments ofthe kit, the thickener component is CMC. In preferred embodiments of thekit, the particulate precursor comprises ASEPTROL S-Tab2 granules andthe thickener component comprises CMC.

Optionally, the kit further comprises an applicator. By the term“applicator,” as the term is used herein, is meant any device including,but not limited to, a dental tray, a syringe, a pipette, a brush, a cup,and the like, suitable for contacting the tooth surface with thecomposition.

Examples

The compositions and methods of use are further described in detail byreference to the following experimental examples. These examples areprovided for purposes of illustration only, and are not intended to belimiting unless otherwise specified. Thus, the compositions, methods ofuse, and kits should in no way be construed as being limited to thefollowing examples, but rather, should be construed to encompass any andall variations which become evident as a result of the teaching providedherein.

Experimental Example 1 Cytotoxicity Analysis

To test the effects of chlorine dioxide on mammalian cells, thefollowing experiment was performed. Two series of samples comprisingdifferent amounts of chlorite anion were prepared. Examples 1-4 used asuper absorbent polyacrylate gel (labeled gel type “S”). Examples 5-8used a carboxymethylcellulose (CMC) gel (labeled gel type “C”).

ASEPTROL S-Tab2 granules were used in the gel compositions used in thisexperiment. The chemical composition of the granules is shown in Table1.

TABLE 1 Component % (wt/wt) Sodium chlorite 7% Dichloroisocyanuric acid,sodium salt 1% Sodium bisulfate 12% Sodium chloride 40% Magnesiumchloride 40%

Sodium chlorite (Aragonesas Energia of Spain) was technical grade,containing nominally 80% (0.8) by weight NaClO₂ and 20% inorganicstabilizer salts such as NaCl, NaOH, Na₂CO₃, and Na₂SO₄.Dichloroisocyanuric acid sodium salt (NaCl₂(CNO)₃.2H₂O) was obtainedfrom Oxychem as ACL-56.

The tablets, from which granules were made, were prepared as essentiallyas described in Example 4 of U.S. Pat. No. 6,432,322, incorporatedherein by reference. In brief, each of the separate components of thegranules was dried. The appropriate quantities of the components wasmixed together and the mixture was compacted into tablet form using ahydraulic table press. The thus-formed tablets were ground into granulesusing a mortar and pestle. The resultant granules were screened using a40 mesh US Standard screen; the −40 mesh size fraction was used in theexperiments.

ASEPTROL S-Tab2 tablets have a high degree of conversion of chloriteanions to ClO₂ (see Examples in U.S. Pat. No. 6,432,322). Typically, asolution made from such tables will contain about 10× as much ClO₂ asresidual chlorite anion. When contacted with water (liquid), the wateris absorbed into the pores of the tablet, where it forms a saturatedaqueous solution of the constituents. Such conditions (highconcentration of chlorite anion and low pH) are advantageous for thereaction of chlorite anion (ClO₂ ⁻) with acid or chlorine to producechlorine dioxide (ClO₂) by reactions:

5NaClO₂+4H+→>4ClO₂+NaCl+4Na⁺+2H₂O   Eq. 3

2NaClO₂+OCl⁻+H⁺→2ClO₂+NaCl+NaOH   Eq. 4

Residual chlorite anion in solution can result from several sources. Oneimportant source of residual chlorite anion in solution is sodiumchlorite, which dissolves from the exterior surface of an ASEPTROLtablet (or granule) into the bulk solution. The conversion rate ofchlorite anion to ClO₂ is low at the very dilute and generallyneutral-pH conditions of the bulk solution, so any chlorite anion thatdissolves from the exterior of a tablet or granule will remainsubstantially unconverted and remain as chlorite anion in solution. As aresult, anything that enhances surface dissolution of sodium chloriteprior to its conversion to ClO₂ will result in an increase in chloriteanion concentration in the resultant solution or gel.

Each base gel (aqueous thickened fluid) was slightly different tocompensate for the different active ingredient concentrations in thefinal samples. The final concentration of thickener component in theprepared gel samples was the same within each series. Each sample wasmade in an about 30 gram amount. The base gels were prepared bycombining deionized water with the gelling agents (thickener component).To allow the gelling agents to become fully hydrated, the mixtures wereallowed to stand for several hours to overnight. The base gel mixtureswere then stirred to homogenize the base gel.

The samples were prepared by combining ASEPTROL granules with a base gelshortly before use. The exposure of the ASEPTROL material to ambienthumidity or water was minimized prior to use to avoid loss of potency.After ASEPTROL granules were added to the base gel, the samples weremixed for 30 seconds with a stainless steel or plastic spatula, cappedand left to stand at room temperature for 5 minutes. The samples werethen mixed a second time for 30 seconds to homogenize the sample.Prepared samples were tightly capped until time of testing. The sodiumchlorite granules and the prepared samples were protected from strong uvlights to limit uv-induced decomposition. Testing was begun no more than2 hours after the samples were prepared.

Chlorine dioxide concentration was assessed by pH 7 buffered titrationusing potassium iodide (KI) and sodium thiosulfate on other samples.Samples 1 and 5 had zero chlorine dioxide. Samples 2 and 6 had about 30ppm ClO₂. Samples 3 and 7 had about 40 ppm and samples 4 and 8 had about580 ppm ClO₂.

There is not an extremely accurate method for measuring directlychlorite anions in a thickened fluid composition. Thus, the maximumconcentration of chlorite anion possibly present in each prepared sampleis provided below. It is expected that the actual amount of chloriteanion is less the maximum, as the reactants are activated in thepresence of an aqueous medium and generate chlorine dioxide, thusconsuming chlorite anions. The maximum amount of chlorite anion possiblypresent in a sample was calculated using the following formula:

-   ((wt. S_tab2 granules×wt. fraction sodium chlorite in granules×wt.    fraction chlorite in sodium chlorite×nominal wt. fraction of sodium    chlorite)×1000)/total wt of final sample. The weight fraction of    sodium chlorite used in S-Tab2 granules is 0.07. The weight fraction    of chlorite in sodium chlorite is 0.74. The nominal weight fraction    of actual sodium chlorite in the sodium chlorite powder (i.e., the    purity of the sodium chlorite) used in the granules is 0.8. Thus,    for instance, the calculation of the milligrams of oxy-chlorine    anion per gram of gel for Ex. 2 is:

((0.143 g.×0.07×0.74×0.8)×1000)/30 grams final sample.

The final formulation for the examples is shown in Tables 2 and 3.

TABLE 2 Component Sample 1 Sample 2 Sample 3 Sample 4 Sodium 1.4 1.4 1.41.4 polyacrylate¹ NaCl 1 1 1 0 Polyethylene oxide² 1.6 1.6 1.6 1.6Deionized water 26 25.9 25.6 25.6 S-Tab2 granules (−40 0 0.143 0.3571.43 mesh) Maximum Mg 0 0.2 0.5 2.0 chlorite per gram gel ¹LUQUASORB1010, BASF Corp ²POLYOX WSR N3000, Dow Chemical Corp.

TABLE 3 Component Sample 5 Sample 6 Sample 7 Sample 8 Sodium 0.75 0.750.73 0.73 carboxymethylcellulose (NaCMC)¹ Na₂HPO₄ 0 0 0 0.2 Deionizedwater 29.3 29.3 29.3 29.2 S-Tab2 granules 0 0.143 0.357 1.43 (−40 mesh)Maximum Mg chlorite 0 0.2 0.5 2.0 per g gel ¹Sigma Aldrich 419338

Each prepared sample was tested in accordance with USP <87>. The methodinvolves determining the biological reactivity of mammalian cellcultures following contact with a topical gel product using an agardiffusion test. The cells in this test are L929 mammalian (mouse)fibroblast cells cultured in serum-supplemented MEM (minimum essentialmedium). A cell monolayer of greater than 80% confluence is grown at 37°C. in a humidified incubator for not less than 24 hours, and is thenoverlaid with agar. The agar layer serves as a “cushion” to protect thecells from mechanical damage, while allowing diffusion of leachablechemicals from the test specimen. Materials to be tested at applied to apiece of filter paper, which is then placed on the agar.

Specifically, a paper disk is dipped in sterile saline to saturate thedisk. The amount of saline absorbed is determined (disk is weighedbefore and after wetting). A quantity of test specimen is dispensed ontothe surface of the wetted disk. The specimen aliquot is kept within theboundaries of the disk but is not spread out over the entire disk. Thedisk with the specimen aliquot is weighed again to assess the amount ofsample on the disk. The disk is then placed on top of the agar overlay.Cultures are evaluated periodically over time for evidence ofcytotoxicity and are graded on a scale of 0 (no signs of cytotoxicity)to 4 (severe cytotoxicity), as summarized in Table 4. A sample is deemedto meet the requirements of the test if none of the cell culture exposedto the sample shows greater than mild cytotoxicity (grade 2) after 48hours of testing. A sample showing grade 3 or 4 reactivity during the 48hours is deemed cytotoxic.

TABLE 4 Grade Reactivity Description of Reactivity Zone 0 None Nodetectable zone around or under specimen 1 Slight Some malformed ordegenerated cells under specimen 2 Mild Zone limited to area underspecimen 3 Moderate Zone extends to 0.5 to 1.0 cm beyond specimen 4Severe Zone extends greater than 1.0 cm beyond specimen

The volume tested of each prepared example in this experimental examplewas about 0.1 cc. The results are shown in Table 5.

TABLE 5 Maximum Gel Mg chlorite Sample # Type per g gel Test result 1 S0 Pass 2 0.2 Pass 3 0.5 Fail 4 2.0 Fail 5 C 0 Pass 6 0.2 Pass 7 0.5 Fail8 2.0 Fail Positive control Fail Negative control Pass

Samples 1, 2, 5, and 6 met the criteria of USP biological reactivity invitro, indicating biocompatibility. Samples 3, 4, 7, and 8 did not meetthe requirements of the USP biological test in vitro. Thus, gels havinga maximum concentration of chlorite anion greater than about 0.2 mgchlorite anion/gram gel produced cytotoxic effect in this experiment.These data suggest that cytotoxicity is related in a dose-dependentmanner to the presence of chlorine dioxide, oxy-chlorine anions or someother constituent(s) of S-TAB2 granules.

Experimental Example 2 Cytotoxicity Analysis

To confirm that cytotoxicity is induced by oxy-chlorine anions and notto other possibly noxious ingredients, the following experiment wasperformed.

A series of samples was prepared to test various ingredients orconditions for their role in inducing cytotoxicity. ASEPTROL S-Tab10tablets were used to prepare some of the samples in this experiment. Thechemical composition of the tablets is shown in Table 6. ASEPTROLS-Tab10 tablets were prepared essentially as described in Example 5 ofU.S. Pat. No. 6,432,322.

TABLE 6 Component % (wt/wt) Sodium chlorite 26% Dichloroisocyanuricacid, sodium salt 7% Sodium bisulfate 26% Sodium chloride 20% Magnesiumchloride 21%

All of the samples comprised NaCMC as the thickener component. Samples9, 16 and 17 were prepared using -40 mesh fraction granules preparedfrom ASEPTROL S-Tab10 tablets. Samples 10, 19 and 20 were prepared usingthe ingredients of ASEPTROL S-Tab10 tablets in a non-granulated form.Specifically, the five ingredients were dried and mixed to form a powderhaving the composition shown in Table 5; the powder was not compactedand granulated. Thus, samples 9 and 10 have identical chemicalcomposition but are made with the solid component in a differentphysical form. Similarly, samples 16 and 19 have identical compositions,as do samples 17 and 20. Samples 11-14 were prepared using a powderhaving a subset of the ingredients in the ASEPTROL tablets, wherein oneor more ingredients was replaced (see second column of Table 7 fordetails). Sample 15 contained substantially pure ClO₂. Sample 18 wasNaCMC alone.

Samples 9-14 and 16-20 were prepared as described in ExperimentalExample 1. In brief, the samples were prepared by combining the solidfraction (e.g., ASEPTROL granules) with a base gel shortly before use.The base gel was NaCMC that was allowed to hydrate. After the solidfraction was added to the base gel, the samples were mixed for 30seconds with a stainless steel or plastic spatula, capped and left tostand at room temperature for 5 minutes. The samples were then mixed asecond time for 30 seconds to homogenize the sample. Prepared sampleswere tightly capped until time of testing. The sodium chlorite granulesand other solid mixture comprising sodium chlorite, and the preparedsamples were protected from strong uv lights to limit uv-induceddecomposition. Testing was begun no more than 2 hours after the sampleswere prepared.

Sample 15 was prepared using a base gel of hydrated NaCMC and asubstantially pure chlorine dioxide solution that was prepared on thesame day the sample was prepared and the test begun. The base gel wasprepared by adding 0.75 gm of sodium carboxymethylcellulose powder(Sigma-Aldrich, 700,000 mole. wt., typ.) to 19.2 gm of deionized water,allowing the mixture to stand in a covered jar for overnight, and mixingto homogenize the base gel. The substantially pure chlorine dioxidesolution was prepared as follows: Twelve (12) ASEPTROL S-Tab10 tablets(1.5 grams each) were placed into 1 liter of potable tap water,producing a deep yellow colored source solution of >1000 ppm chlorinedioxide. Air was bubbled into the bottom of the source solution at arate of about 1 liter per minute to strip chlorine dioxide from thesource solution into the air. The resultant chlorine dioxide-laden airwas then bubbled into the bottom of 1 liter of deionized water to form asolution of pure chlorine dioxide. Only ClO₂ and possibly some watervapor were transferred from the source to the product solution. All thesalt ingredients remained behind in the source solution. As a result,the product solution was a substantially pure solution of ClO₂. Bubblingwas ended when the yellow color of the source solution was nearly gone.A sample of the substantially pure chlorine dioxide solution wasanalyzed for chlorine dioxide concentration using a Hach Model 2010UV/Visible spectrophotometer; the substantially pure solution was foundto contain 700 ppm chlorine dioxide by weight. Ten (10) grams of the 700ppm pure chlorine dioxide solution was added to the base gel and mixedto produce a gel containing about 233 ppm chlorine dioxide andsubstantially no oxy-chlorine anions. As above, the NaClO₂-containingcomponents and the prepared samples were protected from strong UV lightsto limit UV-induced decomposition. All dry solid ingredients wereprotected from water exposure (e.g., ambient humidity) as well.

The samples were tested as described in Experimental Example 1, exceptsamples 17 and 20 were tested at an 0.04 cc dose, rather than an 0.1 ccdose. Testing was begun no more than 2 hours after the samples wereprepared.

The results are shown in Table 7.

TABLE 7 Maximum Mg chlorite Result Sample per gram of USP # final gel<87> 9 Prepared with ASEPTROL S-Tab 10 0.5 Fail granules 10 Preparedwith non-granulated 0.5 Fail ingredients of ASEPTROL S-Tab 10 11 NaDCCAreplaced with cyanuric acid 0.5 Fail 12 NaClO₂ replaced with NaCl 0 Pass13 NaDCCA removed 0.5 Fail 14 NaClO₂ replaced with NaCl, and 0 PassNaDCCA replaced with cyanuric acid 15 Prepared with pure ClO₂ (no othersalts) 0.5 Pass 16 Sample 9 prepared with 3x the water 0.17 Fail 17Sample 9, 0.04 cc dose on disk 0.5 Fail 18 NaCMC alone with no granules,salts or 0 Pass ClO₂ 19 Sample 10 prepared with 3x the water 0.17 Fail20 Sample 10, 0.04 cc dose on disk 0.5 Fail Positive control 0 FailNegative control 0 Pass

Samples 9-11, 13, 16, 17, 19, and 20 all failed to meet the criteria forUSP biological reactivity in vitro. Thus, mimicking the elution-typetest of USP <87> did not alter the results (compare samples 10 and 19,and samples 9 and 16). Reducing the dose did alter the results (comparesample 9 and 17, and samples 10 and 20). These data indicate thatneither the dose used in the test nor the use of gel with 3× the waterplay a role in the observed cytotoxicity.

The results for samples 9 and 10 indicate that the physical form of theASEPTROL component does not noticeably affect the cytotoxicity. Theresults for samples 11 and 13 indicate that the presence of achlorine-producing agent, NaDCCA, does not noticeably affect thecytotoxicity. This result suggests that the observed cytotoxicity doesnot result from chlorine.

Samples 12, 14, 15, and 18 met the criteria for USP biologicalreactivity in vitro, indicating biocompatibility. These data indicatethat the cytotoxicity is not caused by the gellent alone (Sample 18).The observation that Sample 15, which contained pure ClO₂ and no othersalts, did not cause cytopathic effect indicates that chlorine dioxideitself is not the cause of cytotoxicity observed in the samplescomprising ASEPTROL S-Tab10 granules.

The common feature of samples 12, 14, 15 and 18 is that none containchlorite anion. Thus, none of samples 12, 14 and 18 containsoxy-chlorine anions. It is formally possible that sample 15, comprisingpure ClO₂, may contain some oxy-chlorine anions due to the decompositionof ClO₂, however, the amount is insignificant.

In view of these results, it is concluded that oxy-chlorine anions arethe causative agent underlying the cytotoxicity observed in theseexperiments.

Experimental Example 3 Cytotoxicity Analysis

The data in Experimental Example 1 indicate that the cytotoxicity ofoxy-chlorine anions is dose dependent. Specifically, cytotoxicity wasnot observed in gels having a maximum of 0.2 mg chlorite anion per gramgel, whereas cytotoxicity was observed in gels having a maximum of 0.5mg chlorite anion/gr. This experiment was designed to further examinethe cytotoxicity of chlorite anions, using sodium chlorite solution,which permits an more accurate estimate of chlorite anion concentrationin the thickened fluid compositions tested. In addition, thecytotoxicity of a commercially-available over-the-counter,peroxide-based, tooth whitening product, containing 10% hydrogenperoxide as the bleaching agent was also assessed.

Sample 22-24 were prepared by combining an aqueous solution of sodiumchlorite with a base gel shortly before use. Thus, none of samples 22-25contained chlorine dioxide. These samples also did not contain an acidsource or a free halogen source. Samples 22-24 were prepared by mixingthe aqueous sodium chlorite solution with the base gel for 30 seconds,capping the sample and letting it stand at room temperature for 5minutes, the mixing for another 30 seconds. Sample 25 was similarlyprepared but using water instead of a sodium chlorite solution. None ofsamples 22-25 contained an acid source or a free halogen source.

Sample 26 is an over-the-counter (OTC) product that is a gel containing10% hydrogen peroxide; the gel material was used as present on thefoil-wrapped strip.

Sample 21 was prepared using a substantially pure chlorine dioxidesolution prepared by reacting ASEPTROL S-Tab10 tablets into water.Specifically, one 1.5 mg tablet was reacted in 200 ml H₂O. The resultingchlorine dioxide solution was not sparged. Chlorine dioxideconcentration of the solution was about 733 ppm, as assessed using aHach Model 2010 UV-visible light spectrophotometer. Sample 21 thus hadabout 244 ppm ClO₂, after dilution of 1 part solution with 2 parts ofgel

The cytotoxicity results are shown in Table 8.

TABLE 8 Sample # Gel Mg chlorite per gel Result of USP <87> 21 CMC 0Pass (Made with ~700 ppm ClO₂ solution) 22 0.04 Pass 23 1.0 Fail 24 2.0Fail 25 0 Pass 26 unknown OTC product with 10% Fail hydrogen peroxidePositive control Fail Negative control Pass

The results for Samples 22-24 indicate that chlorite anion at elevatedconcentration is cytotoxic to human cells, confirming the conclusionsfrom Experimental Example 2. The result for Sample 21 indicates that ahigh chlorine dioxide concentration thickened fluid composition that isnon-cytotoxic can be prepared using substantially pure chlorine dioxidesolution prepared using ASEPTROL S-Tab10 tablets.

This data also shows that 10% H₂O₂ is cytotoxic (Sample 26) to mammaliancells. Indeed, the reactivity zone extended more than 1 cm beyond thegel specimen, suggesting severe cytotoxicity.

Experimental Example 4 Additional Cytotoxicity Studies

To further examine the relationship between cytotoxicity andoxy-chlorine anion concentration in a thickened fluid composition, thefollowing experiment was performed.

Sample 27-31 were prepared by combining an aqueous solution of sodiumchlorite (10 ml) with 20 g of a base gel (hydrated high viscosity NaCMC)shortly before use. The NaCMC was a USP grade CMC, obtained fromSpectrum Chemical (stock #CA194); a 1% aqueous solution has a viscosityof about 1500-3000 cp. The base gel was prepared using 0.85 g. of NaCMCper 30 g final composition in order to achieve rheology equivalent tothat for the CMC obtained from Sigma Aldrich. None of samples 27-30contained chlorine dioxide. Sample 27 was similarly prepared but usingwater instead of a sodium chlorite solution. Samples 26-30 were preparedby mixing the aqueous sodium chlorite solution (or water) with the basegel until homogenous.

Sample 31, having the same relative composition as Sample 6 and about 40ppm chlorine dioxide, was prepared using a two-syringe mixing method.One syringe contained −40 mesh ASEPTROL S-Tab2 granules (p.048 g). Thesecond syringe contained the base gel (10 grams). The contents of thetwo syringes were combined as follows. The syringe containing thegranules was held with the tip pointing up. The outlet plug was removedand a nylon connector was attached. The other end of the nylon connectorwas attached to the outlet of the syringe containing the base gel. Theplunger of the gel syringe was slowly depressed, expelling the gel intothe granules. The gel-and-granules mixture was then allowed to sit for 5minutes to activate the granules thereby generating chlorine dioxide;the syringes remained connected during this period. After 5 minutes, thesyringe plungers were alternately depressed at a brisk rate to move themixture back and forth between the two syringe bodies at least 15 times,or until the sample was homogenous in color. The gel was then ready foruse the agar diffusion test of USP <87>.

The results of the cytotoxicity testing are shown in Table 9.

TABLE 9 Sample # Gel Mg chlorite per gel Result of USP <87> 27 CMC 0Pass 28 0.1 Pass 29 0.2 Fail 30 0.4 Fail 31 0.2* Pass Positive controlFail Negative control Pass *Maximum amount of chlorite anion possiblypresent; calculated as described in Experimental Example 1

These data further support the discovery that chlorite anion iscytotoxic to human cells in a dose-dependent relationship. Sample 29,which contains 0.2 mg chlorite per gram final composition, failed thetest, whereas Sample 28, which contains 0.1 mg chlorite anion per gr didnot fail. This suggests that chlorine dioxide compositions having lessthan 0.2 mg chlorite anion per gram of composition are not cytotoxic tohuman cells. This outcome also supports the expectation that chloriteanions present in gels made with ASEPTROL granules or powders isconsumed in the generation of chlorine dioxide. Specifically, gelsprepared using ASEPTROL granules or powder and having a maximum possibleamount of 0.2 mg chlorite anion per gram final composition were found tobe non-cytotoxic. Thus, the apparent concentration of chlorite anions inthese gels is estimated to be less than 0.2 mg chlorite per gram.

Experimental Example 5 Tooth Whitening

Tooth whitening efficacy of a chlorine dioxide-containing CMC gel wasassessed in the following experiment. The composition of the gel wassubstantially identical to that of Sample 6 in Table 3, having a maximumpossible chlorite anion concentration of about 0.2 mg/gram final gelcomposition and about 40 ppm ClO₂. The results in Experimental Example 1revealed that this composition is not substantially cytotoxic.

The materials and methods used in this experiment are now described.

Color assessment: Two methods were used to assess tooth shade: 1) visualassessment by Vita Classical Shade Guide; and 2) spectrophotometry.Digital imaging and digital image analysis was also performed to measurecolor of tooth images.

Visual assessment by Vita Classical Shade Guide: Initial baseline shadeand subsequent shade change was assessed by direct comparison to astandard Vita shade guide. The Vita shade guide is arranged in thefollowing order (as recommended by the manufacturer) for valueassessment: B1*A1*B2*D2*A2*C1*C2*D4*A3*D3*B3*A3.5*B4*C3*A4*C4, where B1is the brightest and C4 is the darkest. Two investigators determined theclosest shade match by visualizing the subject tooth against astandardized black background, under controlled, standardizedfluorescent light conditions. In selected whitening experiments,background light conditions were standardized using a hand-held LEDilluminating device at a fixed distance from the test specimen. Theagreement level between operators in shade selection was greater than80%. Disagreements in shade selection were never greater than 1 shadevalue unit (SVU).

Spectrophotometry: A clinical spectrophotometer, Vita EasyShade®(Vident, Brea, Calif.), was used to obtain electronic, quantitative dataabout shade measurement and specific color measurement parameters basedon the CIELAB L*a*b* color space. In this 3D color space system, “L” isthe lightness of an object (ranging from black to white) and is the onlydimension of color that may exist by itself; “a” is a measure of rednessor greenness; and “b” is a measure of yellowness or blueness. The deviceuses a D65 illuminant with a color temperature (in Kelvin) of 6500degrees. At the conclusion of any whitening treatment, ΔL, Δa and Δbvalues were determined and recorded.

Digital images: Digital images of the teeth were taken using an SLRdigital camera/microscope (Olympus DPII Digital Camera/Microscope withOptiva Zoom 100 lens attachment). All images were obtained with standardmanually-entered settings. Approximate fixed lighting of flash apparatuswas configured to provide optimal, standardized imaging conditions.Samples were indexed or oriented repeatedly in a fixed orientation toinsure reproducible image alignment.

Naturally-stained teeth: Human teeth having an intrinsic internalstaining of D4 or lower (i.e., D4 through C4) were used asnaturally-stained teeth. Teeth were sectioned then prepare as describedbelow.

Tea-stained teeth: Human teeth with an intrinsic shade value greaterthan D4 (i.e., B1 through C2) were subjected to tea-based artificialstaining solution as follows. After sectioning the teeth, the exposeddentin surfaces were polished with silicon carbide paper. The dentinsurface was then etched with 37% phosphoric acid etching gel for 20-25seconds, rinsed with water for 30 seconds, and blotted to a moist dentincondition. The teeth were then subjected to continuous staining cycles(by immersion in concentrated tea staining solutions) until the stainintensity appeared unchanged on visual inspection (usually in the Vitashade range of C4-A4).

Tea-stained and naturally-stained teeth were prepared for treatment asfollows. The exposed dentin surfaces of the teeth were coated with threeseparate coats of clear nail polish; each coat was allowed to dry for atleast one hour before the next coat was applied. Teeth were then placedin tap water for at least 24 hours prior to testing. Prior to initiationof treatment, baseline tooth segment shade was assessed by qualitativeand quantitative color assessments. The teeth were mounted inmesio-distal orientation on a glass microscope slide. During treatmentsand between treatments, the teeth were stored in 100% humidity in aplastic bag. During the whitening assay, teeth were removed from theplastic bag, rinsed thoroughly to remove treatment or control whiteningagent, then subjected to qualitative and quantitative color assessment.

Non-cytotoxic ClO₂ gel: The non-cytotoxic ClO₂ aqueous gel materialtested was prepared substantially as described for Sample 6. In brief,thirty (30) grams of aqueous gel base was prepared by adding 0.85 gramsof high viscosity sodium carboxymethylcellulose powder to 29.15 gramsdistilled water. The mixture was allowed to hydrate for at least about 8hours, then was mixed to homogenize the base gel. To the about 30 gramsaqueous NaCMC base gel in a container, 0.143 grams ASEPTROL S-Tab2granules (−40 mesh) was added and mixed gently for 30 seconds. Thecontainer was then tightly capped and the mixture allowed to stand for 5minutes at room temperature. It was then remixed briefly and the ClO₂gel was then ready for use.

The concentrations of most of the constituents of the final gel can becalculated from mass balance or have been measured. The constituents aresummarized in Table 10. The remaining constituents are: about 40 ppmClO₂ by pH 7 titration and less than about 110 ppm un-reacted chloriteanion (ClO₂).

TABLE 10 Chemical species NaCMC Water Na⁺ Mg⁺² Cl− SO₄ ⁻² Cyranuric acidConc., Weight 2.8% 96.7% 0.10% 0.048% 0.26% 0.047% 0.002% %

The prepared ClO₂ gel was a transparent to translucent, light yellow,viscous, pseudoplastic fluid. It had a yield point sufficient to retainits shape when applied in a 1-2 mm layer to teeth, but low enough to besubstantially removed from the tooth surface and soft oral tissue bywiping. The gel was soluble in additional water and can be removed fromthe mouth via rinsing or irrigation. Although ClO₂ is unstable and willslowly decompose over time, the concentration loss over 8 hours underproper storage conditions (kept in closed container, tightly capped orsealed, and minimize exposure to ultraviolet radiation) is less than20%.

Treatment: After mixing, the ClO₂ gel was drawn into a 60 ml plasticsyringe. The 60 ml syringe was used to hold the gel during the assay,and for dispensing gel into a 10 ml plastic syringe. The gel in the 10ml syringe was then dispensed directly onto the tooth section enamelsurfaces as follows. At time zero, about 1 to 1.5 ml gel was dispensedonto the enamel surface of each tooth segment attached to the glassmicroscope slide. The thickness of the resulting gel layer was about 1.5to 3.0 mm in depth. After dispensing the gel onto the tooth segments,the glass slide was placed in a 15 mm×8 mm plastic Ziplocke bag (SCJohnson Co., Racine, Wis.), containing small strips of wet paper towelwithin the bag to maintain 100% humidity in the bag. The paper towelstrips were positioned to eliminate any contact of the plastic bag withthe tooth and gel surfaces.

Upon conclusion of a testing interval (usually 15 minutes), the glassslide was removed from the plastic bag, and the gel was carefullyremoved with an extra soft bristle toothbrush and a gentle stream ofrunning tap water. The tooth segments on the slide were then analyzedfor shade and color, during which time the glass slide was maintainedperiodically in a plastic bag at 100% humidity to avoid undue colorartifact resulting from dehydration.

The gel application procedure was repeated as designed until theexperiment was concluded. The tested tooth segments were stored on theglass microscope slide in 100% humidity for later reference observation.

Naturally-stained and tea-stained human teeth were treated with the ClO₂gel for a total of one (1) hour (4-15 minute consecutive treatments). Asingle batch of ClO₂ gel was used for these consecutive treatments. Forcomparison, other tea-stained teeth were treated with anover-the-counter whitening product containing 10% hydrogen. Treatmentwith the OTC product consisted of 30 minute treatments, in accordancewith the manufacturer's directions. At the end of a treatment, theresidual OTC product left on the tooth after removal of the strip wasremoved from the tooth using a soft bristle toothbrush and a gentlestream of running water. For the multiple day treatments (e.g., 7, 10and 14 days) using the OTC product, typically one 30 minute treatmentoccurred in the morning and the second 30 minute treatment occurred inthe evening.

The Vita Shade values for individual teeth at baseline and after 4×15minute treatments (total of 60 minutes) with ClO₂ gel are tabulated inTables 11 and 12. One of the six tea-stained teeth and one of the sixnaturally-stained teeth each achieved B1 as a result of treatment withClO₂ gel.

TABLE 11 Tea-stained teeth Specimen Baseline shade Post-treatment shadeSVUs T1 A4 C1 9.0 T2 C2 B1 6.0 T3 C4 A3 7.0 T4 A3 A2 9.0 T5 D3 A2 5.0 T6A4 D2 11.0

TABLE 12 Naturally-stained teeth Specimen Baseline shade Post-treatmentshade SVUs N1 A3-D3 D2-A2 >4.0 N2 A3.5-B4 B2 >9.0 N3 A3 B2-A1 >6.0 N4 A3B2-A1 >6.0 N5 A3 B1 8.0 N6 B4 A2 8.0

As shown in FIG. 1, after 30 minutes (2×15 minute treatments) of thenon-cytotoxic ClO₂-containing gel on tea-stained teeth (n=6), the totalshade change was well over 5 Shade Value Units-Vita (SVUs). Fornaturally-stained teeth (n=6), the total shade change was over 6 SVUs.After 45 minutes of treatment (3×15 minutes), the total shade change fornaturally-stained teeth was about 7 SVUs. After one (1) hour (4×15minute) of treatment of tea-stained teeth with the ASEPTROL gel, themean total shade change was 7.83 SVUs. The total shade change fornaturally-stained teeth treated with non-cytotoxic ClO₂ gel was aboutthe same.

The non-cytotoxic ClO₂-containing gel achieved marked lightening ofnaturally-stained teeth after the first 15 minute treatment, andcontinued lightening with continued treatment. In contrast, treatment ofnaturally-stained teeth with the OTC composition containing 10% hydrogenperoxide showed a much lower degree of lightening. There was a modestimprovement (1 SVU) observed after the first 30 minute treatment and nosignificant change after the second 30 minute treatment. Following theOTC product manufacturer's recommended 2×30 minute daily treatments forseven (7) days (total treatment time of 7 hours) resulted in anoticeable improvement (total shade change of 4.9 SVUs); however, theimprovement in tooth whitening was markedly less compared to the ClO₂composition after 4×15 minute consecutive treatments (total treatmenttime of 1 hour). Ten (10) days of treatment (2×30 min per day; totaltreatment time of 10 hours) with the OTC product yielded a total shadechange of 6.1 SVUs. Fourteen (14) days of treatment (total treatmenttime of 14 hours) with the OTC composition containing 10% hydrogenperoxide was needed to result in a total shade change comparable to whatwas achieved with non-cytotoxic ClO₂ gel in one hour of treatment. Thus,the ClO₂-containing gel provided a greater degree of lightening in asignificantly shorter time compared to the hydrogen peroxide-basedcomposition. Furthermore, the ClO₂-containing gel formulation isnon-cytotoxic, whereas the 10% hydrogen-peroxide formulation iscytotoxic (see Experimental Example 3).

The average ΔL value for tea-stained teeth treated for a total of onehour with ClO₂-containing gel was 9.3; the range of values for the 6specimens was 0.8 to 22 ΔL units. The average ΔL value fornaturally-stained teeth treated for a total of one hour withClO₂-containing gel was 8.07. The average change in lightness for teethtreated for a total treatment time of 7 hours with the OTC compositioncontaining 10% hydrogen peroxide was 6.32 ΔL units.

Thus, non-cytotoxic ClO₂-containing gel was extremely effective inbleaching tea-stained teeth and naturally-stained teeth. Notableimprovement in lightening was detected after the first 15 minutetreatment. The degree of lightening achieved after only two-15 minutestreatments with ClO₂-containing gel required 7 days of 2×30 minute dailytreatments with an OTC whitening product containing 10% hydrogenperoxide to achieve. The degree of lightening achieved after four-15minute treatments with ClO₂-containing gel required over 10 days oftreatment with the OTC composition containing 10% hydrogen peroxide toachieve.

Experimental Example 6 Efficacy of ClO₂-Containing Gel Versus 36%Hydrogen Peroxide

This experiment was designed to compare the tooth whitening efficacy ofa non-cytotoxic ClO₂-containing gel with a professional chair-sidewhitening gel containing 36% hydrogen peroxide. This is the highestconcentration of hydrogen peroxide currently in use in professionalchair-side products. The ClO₂-containing gel was prepared as describedin Example 5 and the treatment procedure was the same as in Example 5.

The results are summarized in FIG. 2. After 45 minutes of treatment, thewhitening efficacy of the non-cytotoxic ClO₂-containing gel (˜40 ppmClO₂) approached that of the professional gel. As is well known in theart, gels containing 36% hydrogen peroxide are highly irritating to softtissue in the oral cavity. Thus, achieving comparable tooth whiteningefficacy in the absence of soft tissue irritation is highly desirableand not possible with prior art products.

There are a variety of professional tooth whitening products on themarket which are used by dental professionals. Like the over-the-counterproducts, the professional products are peroxide based. Data for theefficacy of these products is available in the literature (see forinstance Operative Dentistry, 2007, 32-4: 322-327). The literaturevalues suggest that a non-cytotoxic ClO₂-containing gel, a much milderbleaching agent than peroxide, approaches the efficacy of manyperoxide-based professional products and, in some cases, may exceed theefficacy of peroxide-based professional products.

Experimental Example 7 Microhardness of Enamel and Dentin

Hydrogen peroxide is known to adversely affect tooth hard tissues. Toothsensitivity is a common side effect of professional teeth whitenproducts and is believe to originate in morphological changes in theenamel and dentin induced by the high concentration of peroxide. Manyprofessional products recommend the use of sodium fluoride toremineralize the teeth and potassium nitrate to reduce toothsensitivity. To characterize the effect of a non-cytotoxicClO₂-containing gel on enamel and dentin, the microhardness androughness of enamel and dentin was assessed before and after contactwith the chlorine dioxide-containing gel.

The composition of the ClO₂-containing gel is identical to thecomposition in Experimental 5. For both enamel and dentin experimentsusing the ClO₂-containing gel and an over-the-counter product (OTC)containing 10% H₂O₂, total treatment time was 7 hours, consisting of14×30 minute treatments. Multiple batches of ClO₂-containing gel wereprepared and used in this experiment. No batch of ClO₂-containing gelwas used for more than 2 hours. The 7-hour total treatment time is thesame as the treatment time recommended for the OTC product. However,this total treatment time is in great excess of the time needed toachieve tooth whitening with the non-cytotoxic chlorine dioxidecomposition comparable to OTC or professional peroxide-based products(see Examples 5 and 6). For the professional tooth whitening gelcomprising 36% hydrogen peroxide, contact was limited to one hour. Thetooth specimens were stored in tap water prior to, during and aftertreatment.

Microhardness was assessed using a CSM Dynamic Microhardness tester at 2newtons load, 30 seconds load/30 second unloading. Five enamel specimensand five dentin specimens were tested for changes in microhardness. Eachspecimen served as its own control (pre-treatment compared topost-treatment). For enamel hardness, ten measurements per specimen weretaken pre-treatment and post-treatment. Thus, there were 50pre-treatment measurements and 50 post-treatment measurements. Fordentin hardness, five measurements per specimen were taken pre-treatmentand post-treatment, yielding 25 pre-treatment measurements and 25post-treatment measurements. Microhardness data was calculated asVickers hardness values. Statistical analysis consisted of ANOVA(one-way) using an alpha level of 0.05.

Results for enamel hardness (in Vickers hardness values) are shown inTable 13.

TABLE 13 Statistical significant Composition Pre-treatmentPost-treatment p-value (p < 0.05) ClO₂-gel 498.89 ± 70.64  507.40 ±69.92 0.5090 No OTC product 711.57 ± 114.56 722.84 ± 0.8474 No 141.8536% H₂O₂ 538.56 ± 109.30 455.72 ± 36.62 0.000768 YesNeither the ClO₂-containing gel nor the 10% H₂O₂ product induced asignificant change in enamel hardness. In contrast, the professionalproduct induced a statistically-significant reduction (>15% reduction)in enamel hardness. Thus, non-cytotoxic ClO₂-containing gel whitensteeth with an efficacy comparable to that induced by professionalperoxide gels but without adversely affecting enamel hardness.

Results for dentin microhardness (in Vickers hardness values) are shownin Table 14.

TABLE 14 Statistical significant Composition Pre-treatmentPost-treatment p-value (p < 0.05) ClO₂-gel 94.96 ± 9.63 87.65 ± 6.690.0031 Yes OTC product  98.35 ± 15.14 88.71 ± 6.02 0.0118 Yes 36% H₂O₂101.50 ± 21.48  83.45 ± 11.97 0.002212 YesRegarding dentin microhardness, the ClO₂-containing gel induced a minor(7.7%) reduction in dentin microhardness, which was statisticallysignificant. The OTC product (10% peroxide) demonstrated a similar minor(9.8%) reduction in dentin microhardness, which was also statisticallysignificant. Notably, the professional gel induced a dramatic (˜18%)reduction in dentin after only one hour of total contact time.

Thus, non-cytotoxic composition comprising an efficacious amount ofchlorine dioxide for tooth whitening has no statistically significanteffect on enamel microhardness and only a minor effect on dentinmicrohardness. The effect on dentin microhardness is comparable to thatinduced by a commercially-available over-the-counter tooth whiteningproduct.

Experimental Example 8 Surface Roughness of Enamel and Dentin

It has been suggested that increased roughness results in an increasedsurface area that facilitates rebound in whitened teeth. To study theeffect of non-cytotoxic chlorine dioxide containing compositions onsurface roughness, a Surftest 1700 Profilometer was used to assesssurface roughness of enamel and dentin before and after treatment withvarious whitening compositions.

Four enamel specimens and four dentin specimens were tested. Eachspecimen served as its own control. Twelve measurements per specimenwere taken pre-treatment and another twelve per specimen were takenpost-treatment. Contact time for the ClO₂-containing gel was a total of2.5 hours, consisting of 4-15 minute treatments and 3-30 minutetreatments. Additional 30 minute treatments with ClO₂-containing gelwere performed until a total treatment time of 7 hours was achieved. Thespecimens were stored in tap water prior to, during, and aftertreatment. The non-cytotoxic chlorine dioxide-containing composition isthe same as that used in Experimental Example 8. An over-the-counter(OTC) whitening product containing 10% hydrogen peroxide was alsotested, in 14-30 minute consecutive treatments. A professional toothwhitening gel containing 36% hydrogen peroxide was also tested. For theprofessional tooth whitening gel, contact was limited to one hour.Statistical analysis consisted of ANOVA (one-way) using an alpha levelof 0.05.

The profilometry data for the non-cytotoxic chlorine dioxide compositionand the OTC product containing 10% hydrogen peroxide are shown in Table15.

TABLE 15 R_(a) values ANOVA Pre- Post-treatment p-value treatment 2.5hrs 7 hrs 2.5 hrs 7 hrs Enamel OTC 0.037692 ± 0.00914  N/D 0.048742 ±0.0157  N/D 0.04674* ClO₂ 0.024975 ± 0.002445 0.021508 ± 0.000888 0.02295 ± 0.000666 0.005799* 0.048636* Dentin OTC 0.032053 ± 0.007332N/D 0.051742 ± 0.00882 N/D 0.0000053* ClO₂  0.03998 ± 0.005542 0.03775 ±0.00466 0.044608 ± 0.00392 0.296884 0.027545* N/D = not determined.*Statistically significant

The average surface roughness, Ra, of enamel before treatment with thenon-cytotoxic chlorine dioxide composition was about 0.025 and 0.021after 2.5 hours of treatment, and about 0.023 after 7 hours oftreatment. Thus, no adverse effect by the ClO₂-containing gel wasdetected on enamel average surface roughness, even despite extendedduration of contact. Indeed, enamel average surface roughness wasactually smoother by about 13-14% after 2.5 hours of ClO₂-containing geltreatment in this experiment, indicating an enamel polishing effectunexpected in the absence of an abrasive. Furthermore, the effect ondentin average surface roughness after 2.5 hours of treatment wasstatistically insignificant. After 7 hours of treatment, the increase indentin average surface roughness was only about 8%. As previously noted,7 hours of treatment is in excess of the time need to achieve toothwhitening with this composition comparable to OTC or professionalperoxide-based products (see Examples 5 and 6). Thus, the non-cytotoxicchlorine dioxide composition can produce tooth whitening comparable toOTC or professional products without substantial damage to enamel ordentin surface roughness.

In contrast, OTC product-treated teeth after the recommended 7 hours ofcontact time showed an increase of enamel surface roughness of greaterthan about 29%. The increase in dentin surface roughness after 7 hourswas greater than 60%. After only 1 hour of treatment, the professionalperoxide product, containing 36% hydrogen peroxide, increased surfaceroughness of both the enamel and the dentin by about 203%.

Regarding effect on enamel, there is little evidence of surfacemorphology alteration in enamel is observed in the specimen (FIG. 3B)treated with non-cytotoxic ClO₂-containing gel for 7 hours, compared tothe non-treated control tooth (FIG. 3A). Indeed, the fine-finishingscratches (result of tooth specimen preparation) are still evident. Incontrast, the photograph of the specimen (FIG. 3C) treated with the 36%peroxide gel for one hour reveals significant areas of enamel alterationand possible erosion.

As shown in FIG. 4A, the dentin surface morphology of a control(untreated) tooth has some dentinal tubules exposed or revealed, withother dentinal tubules hidden by a dentin smear layer. Some smear plugsare evident within exposed tubules. The surface of the representativespecimen treated with an OTC product containing 10% H₂O₂ (FIG. 4B)reveals a greater number of dentinal tubules exposed, compared withuntreated control dentin surface. Exposed tubules appear somewhatenlarged compared to control surface, and many tubules appear openwithout the presence of occluding smear plugs. The representative toothspecimen (FIG. 4C) treated with non-cytotoxic ClO₂-containing gel has agreater number of dentinal tubules exposed or revealed, compared tocontrol surface; but fewer in number and smaller in dimension than thetubules present in the OTC-treated specimen. Exposed tubules are presentas narrow “slits” with limited openings; some dentinal tubules arehidden by an apparent smear layer; some smear plugs evident withinexposed tubules. Thus, the surface of the tooth treated withnon-cytotoxic ClO₂-containing gel more closely resembles the surface ofthe control tooth.

While not wishing to be bound by theory, it is believed that thealteration in dentin surface induced by the 10% hydrogen peroxide gel,and therefore expected for the higher concentration professionalproducts, may underlie at least in part the tooth sensitivity issuecommon in over-the-counter and professional peroxide whitening products.

Experimental Example 9 Mixed Agent Compositions

The following experiment was performed to assess cytotoxicity ofhydrogen peroxide solutions in the presence or absence of 200 ppmchlorine dioxide.

A 600 ppm ClO₂ stock solution was prepared by dissolving an 80 mgASPETROL S-Tab2 tablet in 3.3 grams of water. Samples were prepared bymixing 6.3 grams of appropriate dilutions of hydrogen peroxide (3% w/v)Topical Solution USP with either 3.38 grams of 600 ppm ClO₂ solution(samples 35-37) or 3.3 grams of water (samples 32-34).

Cytotoxicity was assessed in accordance with USP <87>as describedpreviously. The 600 ppm ClO₂ stock solution and hydrogen peroxidedilutions were mixed immediately prior to applying the mixture to theagar.

The data are presented in Table 16.

TABLE 16 H₂O₂ ClO₂ Conc. (% Conc. 6 24 48 Test Sample w/v) (ppm) Initialhours Hours Hours Results 32 2 0 0 3 3 3 Fail 33 1 0 0 3 3 3 Fail 34 0.10 0 3 3 3 Fail 35 2 200 0 3 3 3 Fail 36 1 200 0 3 3 3 Fail 37 0.1 200 03 3 3 Fail

The data show that for the concentrations tested, there was nodifference in cytotoxicity observed. In other words, the addition of 200ppm chlorine dioxide to hydrogen peroxide solutions did not alter thedegree of cytotoxicity observed for the hydrogen peroxide. Since thebleaching efficacy due to the added chlorine dioxide would be expectedto increase the bleaching efficacy of the mixed agent composition overthe peroxide composition containing the same amount of peroxide, thebleaching efficacy of the mixed agent composition would be expected tobe comparable to a peroxide composition containing a greaterconcentration of peroxide than in the mixed agent composition.Therefore, these data suggest that the cytotoxicity of a bleachingcomposition comprising hydrogen peroxide and chlorine dioxide can belower in cytotoxicity compared to a composition comprising no chlorinedioxide and a sufficient quantity of hydrogen peroxide to havecomparable bleaching efficacy as the mixed agent composition.

Experimental Example 10 Clinical Trial

The purpose of this human subject feasibility study is to evaluate theefficacy of a chair-side, 1 hour (4 separate 15 minute treatments),in-office application of a non-cytotoxic tooth whitening compositioncomprising about 40 ppm chlorine dioxide and high viscosity sodium CMCas the thickener component. The composition will comprise no more thanabout 0.2 mg per gram composition oxy-chlorine anions. Shade change andtooth sensitivity to the tooth whitening agent, as well as patientresponse to the treatment, will be evaluated. Fifteen subjects will beenrolled in a clinical trial. Subjects will receive a 1 hour (4 separate15-minute treatments), in-office treatment with the experimental toothwhitening agent in this pilot, single-arm, non-controlled, prospective,case-controlled study. All subjects will be monitored at baseline,immediately post-application of the in-office treatment, 72 hourspost-application, and one week post application of the in-officetreatment. Trained examiners using Vita (Vita Zahnfabrik) shade guidesand color transparencies will monitor color changes. The Vita shadeguide is one of the acceptable evaluation methods included in the ADAguidelines. The transparencies will be used as a record of colorchanges. Tooth sensitivity will be monitored using a standardized scalefor the patient to mark at baseline, immediately post in officetreatment, 72 hours post in office treatment and one week post in officetreatment.

Subjects will be selected on the basis of having maxillary anteriorteeth that are shade Vita A3 or darker, as judged by comparison with avalue-oriented Vita shade guide. Subjects must be 18-65 years old, havegood general health, and have good dental health and oral hygiene.Patients with active caries, periodontal disease, large anterior crownsor restorations, previously-bleached or tetracycline-stained teeth willbe excluded from the study.

Upon acceptance into the study, each patient will be examined by one ofthe clinicians. A baseline Vita shade will be determined by twoevaluators; the consensus shade of the six test maxillary anterior teethand the six control mandibular teeth will be recorded by the dentist. Adigital color transparency (Photomed S1 Pro Digital Clinical Camera—FujiBody; Sigma Lens; Nikon Flash) will be made at a 1:1 magnification. Thematching Vita shade tab will be included in the photograph.

An alginate impression of the maxillary arch will be made and poured indental stone. Custom whitening trays will be fabricated for each patientusing the material and design recommended by the manufacturer. Allsubjects will receive a prophylaxis before starting treatment and willbe asked to mark a standardized scale to rate baseline sensitivity.

The clinical trial has been initiated, and preliminary data has beenobtained.

Experimental Example 11 Mixed Agent Bleaching Composition Study

An experiment is performed to study the tooth whitening efficacy of amixed agent bleaching composition. The mixed agent composition (testarticle) is an aqueous thickened fluid and comprises 40 ppm chlorinedioxide and 10% hydrogen peroxide. Two control compositions are alsotested. The first control (C1) is an aqueous thickened fluid andcomprises 40 ppm chlorine dioxide. The second control (C2) is an aqueousthickened fluid and comprises 10% hydrogen peroxide. The experiment isperformed substantially like Experimental Example 5. Additional data tobe collected include one or more of enamel microhardness, dentinmicrohardness, enamel surface roughness and dentin surface roughness, asdescribed in Experimental Examples 7 and 8.

The results are expected to reveal that the test article has a toothwhitening efficacy at least as good as either of C1 and C2. Further, itis contemplated that the tooth whitening efficacy of the test article isgreater than the sum of the efficacies of C1 and C2. That is, the toothwhitening efficacy of the mixed agent bleaching composition iscontemplated to be synergistic with respect to the efficacies of theindividual bleaching agents.

It is further contemplated that the mixed agent bleaching compositioncauses or is associated with hard tooth tissue damage about equal orless than that observed for C2. In addition, the mixed bleaching agentcauses or is associated with irritation to soft oral tissue about equalto or less than that observed for C2.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety for all purposes.

While the compositions, kits, and methods have been disclosed withreference to specific embodiments, it is apparent that other embodimentsand variations of the compositions, kits, and methods may be devised byothers skilled in the art without departing from the true spirit andscope of the these compositions, kits, and methods. The appended claimsare intended to be construed to include all such embodiments andequivalent variations.

1. A bleaching composition for whitening a tooth surface comprisingchlorine dioxide and at least a second bleaching agent, wherein: i) thecytotoxicity of the bleaching composition is about equal to or less thanthe cytotoxicity of a composition comprising no chlorine dioxide and asufficient quantity of the at least second bleaching agent to havecomparable bleaching efficacy; ii) the bleaching composition isassociated with soft oral tissue irritation about equal to or lessirritation than associated with a composition comprising no chlorinedioxide and a sufficient quantity of the at least second bleaching agentto have comparable bleaching efficacy; or iii) the bleaching compositionis associated with hard tooth tissue damage about equal to or less thanthe damage associated with a composition comprising no chlorine dioxideand a sufficient quantity of the at least second bleaching agent to havecomparable bleaching efficacy.
 2. The composition of claim 1, furthercomprising a thickener component.
 3. The composition of claim 1, whereinthe composition comprises about 5 to about 1000 ppm chlorine dioxide. 4.The composition of claim 1, wherein the composition comprises about 30to about 40 ppm chlorine dioxide.
 5. The composition of claim 1, whereinthe composition comprises less than about 0.2 milligrams oxy-chlorineanion per gram composition.
 6. The composition of claim 1, wherein thecomposition has a pH from about 4.5 to about
 11. 7. The composition ofclaim 1, wherein the composition has a pH from about 5 to about
 9. 8.The composition of claim 1, wherein the composition has a pH greaterthan about 6 and less than about
 8. 9. The composition of claim 1,wherein the pH is equal to or less than about pH 5 and the compositionfurther comprises a remineralizing agent.
 10. The composition of claim9, wherein the composition further comprises a desensitizing agent. 11.The composition of claim 1, wherein the thickener component is selectedfrom the group consisting of natural hydrocolloids, semisynthetichydrocolloids, synthetic hydrocolloids, and clay.
 12. The composition ofclaim 1 1, wherein the thickener component is a semisynthetichydrocolloid.
 13. The composition of claim 12, wherein the semisynthetichydrocolloid is carboxymethylcellulose.
 14. The composition of claim 1,wherein the at least second bleaching agent is selected from the groupconsisting of: alkali metal percarbonates, carbamide peroxide, sodiumperborate, potassium persulfate, calcium peroxide, zinc peroxide,magnesium peroxide, hydrogen peroxide complexes, hydrogen peroxide, andcombinations thereof.
 15. The composition of claim 1, wherein thecomposition has a bleaching efficacy and wherein the bleaching efficacyis synergistic relative to the efficacies of the individual bleachingagents.
 16. A method of whitening a tooth surface, the method comprisingcontacting the surface of a tooth with an efficacious amount of ableaching composition as recited in claim
 1. 17. The method of claim 16,wherein the composition comprises about 5 to about 1000 ppm chlorinedioxide.
 18. The method of claim 16, wherein the composition comprisesless than about 0.2 milligrams oxy-chlorine anion per gram composition.19. The method of claim 16, wherein the composition has a pH equal to orless than about pH 5 and the composition further comprises aremineralizing agent.
 20. The method of claim 16, wherein the contactedtooth surface is whitened by at least about 3 shade value units.